Are interphylum spiralian relationships resolvable?
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This important study probes the long-standing failure to resolve evolutionary relationships between the classical "spiralian" taxa-i.e., annelids, molluscs, brachiopods, platyhelminths and nemerteans-and provides convincing evidence that the branches leading to them are so short as to be unreliable guides to their relationships. This, in turn, has wide-ranging implications for our understanding of animal body plan evolution and the interpretation of early animal fossils.
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Abstract
The phyla making up the major animal clade of Spiralia have been clear since the advent of molecular phylogenetics; the relationships between these spiralian phyla have not. The lack of consensus over the relationships between these important animal phyla might be a clue implying their emergence in an explosive radiation. Focussing on the five largest spiralian clades (Annelida, Brachiozoa, Mollusca, Nemertea and Platyhelminthes) and using two phylogenomic datasets, we have applied site-bootstrapping and taxon-jackknifing to explore this example of taxonomic instability. Analyses on the 105 possible rooted trees relating them showed that interphylum branches are very short. Preference for rooting Spiralia on Platyhelminthes is enhanced by a long-branch artefact. Most analyses on the 15 unrooted trees showed a preference for the same topology but the support for this tree over other solutions was not significant. We conclude that the spiralian phyla emerged in rapid succession resulting in a difficult to resolve radiation. The deep history we infer for Spiralia has wide ranging implications for our interpretation of Cambrian fossils and for the evolution of traits such as biomineralization, segmentation and larvae.
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eLife Assessment
This important study probes the long-standing failure to resolve evolutionary relationships between the classical "spiralian" taxa-i.e., annelids, molluscs, brachiopods, platyhelminths and nemerteans-and provides convincing evidence that the branches leading to them are so short as to be unreliable guides to their relationships. This, in turn, has wide-ranging implications for our understanding of animal body plan evolution and the interpretation of early animal fossils.
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Reviewer #1 (Public review):
[Editors' note: this version has been assessed by the Reviewing Editor without further input from the original reviewers. The revised version adequately addresses the relatively minor comments from the previous round of review.]
Summary:
This interesting paper probes the problematic relationships between the classical "spiralian" taxa, i.e., annelids, molluscs, brachiopods, platyhelminths and nemerteans, and shows that the branches leading to them are so short as to be unreliable guides to their relationships. This, in turn, has important implications for how we view the origin of the animal phyla.
Strengths:
A very careful analysis of a famous old problem with quite significant results. The results seem to be robust and support their conclusions.
It often passes uncommented that many different trees are …
Reviewer #1 (Public review):
[Editors' note: this version has been assessed by the Reviewing Editor without further input from the original reviewers. The revised version adequately addresses the relatively minor comments from the previous round of review.]
Summary:
This interesting paper probes the problematic relationships between the classical "spiralian" taxa, i.e., annelids, molluscs, brachiopods, platyhelminths and nemerteans, and shows that the branches leading to them are so short as to be unreliable guides to their relationships. This, in turn, has important implications for how we view the origin of the animal phyla.
Strengths:
A very careful analysis of a famous old problem with quite significant results. The results seem to be robust and support their conclusions.
It often passes uncommented that many different trees are published about animal relationships, yet some parts of the tree seem extremely difficult to resolve; the spiralians are perhaps the most difficult case. More recently, problems about sponges or ctenophores as sister groups to the rest of the animals have alerted us to major areas of uncertainty in large-scale phylogenetic reconstruction; this paper is a welcome reminder that other, perhaps even harder, problems exist which may be difficult to ever resolve with the (molecular) data we have.
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Reviewer #2 (Public review):
Summary:
The relationships among the phyla making up Spiralia - a major clade of animals including molluscs, annelids, flatworms, nemerteans and brachiopods - have been challenging from a phylogenomic perspective despite decades of molecular phylogenetic effort. Every topology uniting subsets of these phyla has been recovered with apparent support in at least one study, yet no consensus has emerged even from large-scale genomic datasets. Serra Silva and Telford set out to determine whether this instability reflects a genuine biological signal being obscured by analytical limitations, or whether it reflects a rapid, near-simultaneous origin of these phyla that has left behind in modern genomes far too little phylogenetic information to resolve. They focused deliberately on five phyla, reducing the problem to …
Reviewer #2 (Public review):
Summary:
The relationships among the phyla making up Spiralia - a major clade of animals including molluscs, annelids, flatworms, nemerteans and brachiopods - have been challenging from a phylogenomic perspective despite decades of molecular phylogenetic effort. Every topology uniting subsets of these phyla has been recovered with apparent support in at least one study, yet no consensus has emerged even from large-scale genomic datasets. Serra Silva and Telford set out to determine whether this instability reflects a genuine biological signal being obscured by analytical limitations, or whether it reflects a rapid, near-simultaneous origin of these phyla that has left behind in modern genomes far too little phylogenetic information to resolve. They focused deliberately on five phyla, reducing the problem to a tractable set of 15 unrooted and 105 rooted topologies, and applied a suite of complementary approaches across two independent datasets and multiple substitution models to test whether any topology is significantly preferred over alternatives.
Strengths:
(1) The conceptual framing of the problem is excellent, and the study makes a convincing case across several lines of evidence. By enumerating all possible topologies and demonstrating empirically that every one of the 15 unrooted arrangements has been recovered as the preferred solution in at least one published study, the authors make a strong argument about the state of the field. The use of two entirely independent datasets as a consistency check is great, and convergence between them, where it occur,s substantially strengthens confidence in the conclusions.
(2) It is my view that the simulation framework is a particular strength. Generating data on a fully unresolved star tree and scoring those data under both correctly-specified and misspecified substitution models provides convincing evidence that the strong preference for rooting Spiralia on the flatworm branch is, at least partly, an analytical artefact driven by the exceptionally long branch in combination with compositional heterogeneity across sites. This is an important methodological demonstration with implications beyond spiralian phylogenetics, as the same issue is likely to affect other deep, long-branched lineages in the animal tree of life.
(3) The randomised taxon-jackknifing approach is a very nice addition here. The demonstration that preferred topologies shift depending on which species happen to be sampled (even within the same phylum) is a convincing indicator of weak signal, and provides a practical caution for future studies that may report strong support for a particular spiralian arrangement based on a fixed taxon sample.
(4) The branch-length analyses, benchmarking internal interphylum branches against the already disputed and extremely short branch uniting deuterostomes (work also by this group), are well-conceived and solid.
(5) I think it is worth highlighting the notable intellectual honesty throughout the paper: the authors do not overstate their results, correctly acknowledging that while the unrooted topology grouping molluscs with brachiopods and flatworms with nemerteans emerges most consistently, this preference is not statistically significant under more adequate substitution models and may itself carry some artefactual component.
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Author response:
The following is the authors’ response to the original reviews.
Public Reviews:
Reviewer #1 (Public review):
Summary:
This interesting paper probes the problematic relationships between the classical "spiralian" taxa, i.e., annelids, molluscs, brachiopods, platyhelminths and nemerteans, and shows that the branches leading to them are so short as to be unreliable guides to their relationships. This, in turn, has important implications for how we view the origin of the animal phyla.
Strengths:
A very careful analysis of a famous old problem with quite significant results. The results seem to be robust and support their conclusions.
It often passes uncommented that many different trees are published about animal relationships, yet some parts of the tree seem extremely difficult to resolve; the spiralians are perhaps the …
Author response:
The following is the authors’ response to the original reviews.
Public Reviews:
Reviewer #1 (Public review):
Summary:
This interesting paper probes the problematic relationships between the classical "spiralian" taxa, i.e., annelids, molluscs, brachiopods, platyhelminths and nemerteans, and shows that the branches leading to them are so short as to be unreliable guides to their relationships. This, in turn, has important implications for how we view the origin of the animal phyla.
Strengths:
A very careful analysis of a famous old problem with quite significant results. The results seem to be robust and support their conclusions.
It often passes uncommented that many different trees are published about animal relationships, yet some parts of the tree seem extremely difficult to resolve; the spiralians are perhaps the most difficult case. More recently, problems about sponges or ctenophores as sister groups to the rest of the animals have alerted us to major areas of uncertainty in large-scale phylogenetic reconstruction; this paper is a welcome reminder that other, perhaps even harder, problems exist which may be difficult to ever resolve with the (molecular) data we have.
Weaknesses:
The paper could have perhaps drawn out some of the implications of its results in a clearer manner.
Reviewer #2 (Public review):
Summary:
The relationships among the phyla making up Spiralia - a major clade of animals including molluscs, annelids, flatworms, nemerteans and brachiopods - have been challenging from a phylogenomic perspective despite decades of molecular phylogenetic effort. Every topology uniting subsets of these phyla has been recovered with apparent support in at least one study, yet no consensus has emerged even from large-scale genomic datasets. Serra Silva and Telford set out to determine whether this instability reflects a genuine biological signal being obscured by analytical limitations, or whether it reflects a rapid, near-simultaneous origin of these phyla that has left behind in modern genomes far too little phylogenetic information to resolve. They focused deliberately on five phyla, reducing the problem to a tractable set of 15 unrooted and 105 rooted topologies, and applied a suite of complementary approaches across two independent datasets and multiple substitution models to test whether any topology is significantly preferred over alternatives.
Strengths:
(1) The conceptual framing of the problem is excellent, and the study makes a convincing case across several lines of evidence. By enumerating all possible topologies and demonstrating empirically that every one of the 15 unrooted arrangements has been recovered as the preferred solution in at least one published study, the authors make a strong argument about the state of the field. The use of two entirely independent datasets as a consistency check is great, and convergence between them, where it occur,s substantially strengthens confidence in the conclusions.
(2) It is my view that the simulation framework is a particular strength. Generating data on a fully unresolved star tree and scoring those data under both correctly-specified and misspecified substitution models provides convincing evidence that the strong preference for rooting Spiralia on the flatworm branch is, at least partly, an analytical artefact driven by the exceptionally long branch in combination with compositional heterogeneity across sites. This is an important methodological demonstration with implications beyond spiralian phylogenetics, as the same issue is likely to affect other deep, long-branched lineages in the animal tree of life.
(3) The randomised taxon-jackknifing approach is a very nice addition here. The demonstration that preferred topologies shift depending on which species happen to be sampled (even within the same phylum) is a convincing indicator of weak signal, and provides a practical caution for future studies that may report strong support for a particular spiralian arrangement based on a fixed taxon sample.
(4) The branch-length analyses, benchmarking internal interphylum branches against the already disputed and extremely short branch uniting deuterostomes (work also by this group), are well-conceived and solid.
(5) I think it is worth highlighting the notable intellectual honesty throughout the paper: the authors do not overstate their results, correctly acknowledging that while the unrooted topology grouping molluscs with brachiopods and flatworms with nemerteans emerges most consistently, this preference is not statistically significant under more adequate substitution models and may itself carry some artefactual component.
Weaknesses:
(1) The restriction to five phyla is the most significant limitation, as the authors acknowledge this and give a clear computational justification, but readers should be aware that the paper's convincing conclusions apply specifically to the five focal phyla and the evidence remains incomplete with respect to spiralian phylogeny as a whole.
(2) The treatment of substitution model adequacy, while commendably thorough for site-heterogeneous models, is necessarily bounded. The authors note that models accounting for non-stationarity, across-lineage compositional heterogeneity, or mixtures of tree histories might yield different results, and that even the most sophisticated currently available approaches have not produced consistent spiralian topologies across studies. This is not a criticism of what has been done here - the analytical scope is reasonable and well-implemented - but it means the paper cannot be read as a definitive demonstration that no model will ever resolve these relationships. The distinction between a true hard polytomy and a radiation that is effectively unresolvable given current data and methods could be drawn more sharply in the discussion.
(3) The reticulation-aware coalescent analyses are presented somewhat briefly relative to the likelihood-based topology scoring. The finding that flatworms are recovered within a paraphyletic jaw-bearing animal clade in both summary trees - interpreted as long-branch attraction - is striking, and its implications for gene-tree-based approaches to spiralian rooting deserve more discussion than they currently receive.
(4) The central conclusions - that interphylum branches in Spiralia are extraordinarily short, that topological preferences are strongly model-dependent and taxon-sampling-sensitive, and that an ancient rapid radiation is the most parsimonious explanation - are convincingly supported by the evidence presented. The identification of flatworm long-branch attraction as an important confounding factor in rooting analyses is itself an important and well-demonstrated result.
Conclusion:
This paper clearly makes an important contribution to the ongoing debate about spiralian relationships and, more broadly, to methodological discussions about how to handle anciently diversified clades where phylogenetic signal is genuinely limited. The exhaustive topology-scoring framework combined with taxon-jackknifing and simulation under unresolved trees is a valuable methodological template that could usefully be applied to other notoriously difficult nodes in the animal tree. I thoroughly enjoyed the discussion of the implications of these findings for interpreting Cambrian fossils and the evolutionary history of shells, segmentation, larval types and other characters - it is both thoughtful and thought-provoking and will be of broad interest well beyond the phylogenomics and zoology communities. From a very practical perspective, the data and scripts provided make the work useful to researchers wishing to apply similar approaches to other groups.
Reviewer #3 (Public review):
Summary:
This paper addresses the controversial internal relationships within the Spiralia, a major clade of invertebrate animals including molluscs, annelids, brachiopods and flatworms.
Strengths:
Performs a range of empirical analyses and simulations that address the core question. Although a favoured unrooted topology finds some support, this is not strongly endorsed in the paper.
Weaknesses:
(1) Only considers a subset of relevant phyla (e.g. gastrotrichs are relevant to the phylogenetic position of Platyhelminthes), although how this would change the scale of the analyses (i.e. number of topologies) is addressed in the paper.
(2) Discussion of Spiralia evolution and broader context, particularly the relevance for the fossil record. Line 448: our current understanding of the early spiralian fossil record is quite consistent with the main results of this paper. For example, there are very few claims for fossils that sit on the short branch leading to Spiralia (or Lophotrochozoa as defined here) that this paper discusses. Many of the key fossils that inform on the characters discussed in the introduction, which have unusual character combinations, have an apomorphy of one of the phyla discussed, and so are resolved as members of the stem lineages of particular phyla.
(3) This is what you would expect with long phylum stem lineages (line 148) and a short spiralia stem lineage. For example, the mollusc Wiwaxia has chaetae, but a mollusc like Radula (Smith 2012), the conchiferan mollusc Pelagiella has chaetae and a coiled shell (Thomas et al. 2020). The only fossil groups that are routinely discussed as belonging to the stem lineage of more than one phylum are the tommotiids, which have chaetae, segmentation and a complex mineralised skeleton (but not shells in the brachiopod/mollusc sense, see Guo et al 2023) but they sit on the lophophorate stem lineage, a synapomorphy rich group the monophyly of which the present paper endorses (e.g. line 435). The fossil record is consistent with the scenario presented in line 442, e.g. convergent loss or reduction of chaetae and segmentation and convergent evolution of shells in molluscs and brachiopods.
We thank the reviewers for their kind comments. Please see below for detailed responses to all identified weaknesses.
Recommendations for the authors:
Reviewer #1 (Recommendations for the authors):
Some minor comments that might help improve the paper:
(1) Abstract L17. "Most analyses on the 15 unrooted trees showed a preference for the same topology but the support over other solutions was non significant" - I don't really understand this sentence in the context of the paper; it makes it sound as if the tree is, after all, well resolved! Non-significant, or not significant better than non significant?
Having read the rest of the paper I see what this refers to (uT4), but still I don't understand the second clause.
Re-written to clarify.
(2) Introduction L31. This makes it sound as if phoronids are actually part of brachiopods, and while that was recovered by Cohen and Weydmann 2005, I'm not sure if it's really a general result. In addition, rather than using "brachiopods plus phoronids" everywhere, you could use "Brachiozoa" (Cavalier-Smith 1998, Biol. Rev).
We have updated our text and figures to use Brachiozoa.
(3) L36-37. Yes, but the presence of Chaetagnatha in this clade is suggestive that their primitive body size is not small.
Have made clear that chaetognaths are not all tiny.
(4) L85. Kumar et al. may have claimed that Spiralia are as old as 670, but many other analyses would suggest a range of different results. Why choose just this one? In addition, this age seems rather incompatible with your results.
We agree this maximum age is highly improbable (the principal point remains the deep age of the protostomes). We have used a different reference and refer to a generally acceptable minimum age only.
(5) L88. The key part of this sentence, "proving a hard polytomy", comes at the end of a long set of references that makes it hard to connect to the lead-in "given the age of", so I would suggest rephrasing.
Rephrased for clarity.
(6) L109. It is unclear what this means in the context: "and even support multiple topologies".
Re-worded for clarity.
(7) Figure 1. Why did you choose to indicate brachiopods plus phoronids as a larval form, unlike the other clades? Perhaps it's because we don't know what the last common ancestor of the two looked like (unless P is an ingroup of B), but that's arguably true for some of the other clades as well!
Apologies, this was laziness as we already had a line drawing of an actinotroch larva. Have improved the images in figures 1 and 5 where required.
(8) L164. Reticulation-aware analyses. As I understand it, this would include introgression, hybridization, etc. However, incomplete lineage sorting has also been invoked, not just for Cambrian-explosion age events but also for other major radiations, such as for angiosperms and birds. How significant might ILS be for generating the results you get?
Section title amended. Results section updated to reflect this. We now explicitly mention the potential impact of ILS and introgression on spiralian relationships in our discussion.
Unrooted trees analysis:
(9) L405 on. Maybe it would be worth including a figure showing the relative branch lengths of uT4. All the images of trees show similar-length branches, which gives off the wrong impression within the context of the paper!
We understand the motivation, but we worry that showing uT4 as the sole phylogram may end up with this being interpreted by a casual reader as being the main result of the paper. Hopefully the figures with branch lengths encompass this information well enough and with no danger of misinterpretation.
(10) L430 on. Why is this a "conservative" interpretation?
Yes agreed not clear. Have changed to “We interpret our results as showing that…”
(11) You mention synapomorphy accumulation time and implicitly equate shortness of branches with shortness of time. However, other options are available under varying diversification rate models (e.g. ClaDs, Barido-Sottani et al. 2023 Syst. Biol.; CET, Budd and Mann 2025, Syst.Biol.). In particular, the latter paper shows that when unusually large clades are selected for study (as is arguably the case here), then those clades are likely to have started with very high "evolutionary tempo", which speeds up all aspects of evolution, including diversification rates.
In the Budd and Mann scenario large clades begin with high tempo of cladogenesis, high substitution rate and high diversification rate (rapid origin of new characters). This would suggest that the period of the radiation was extra rapid (even less time than in a ‘normal’ period during which smaller clades emerge) so we feel the point stands.
(12) L449. Maybe refer to the Song et al. paper again here on scaphopods plus bivalves, as it makes the same sort of points, albeit in a slightly different context.
We thank the reviewer for the suggestion and have added the citation where relevant.
(13) Finally, to return to L20. You mention implications for the Cambrian fossil record, but then fail to deliver any!
We have hopefully addressed this remark in the discussion better (at least to the extent we are qualified to).
Yet if you are correct, then synapomorphy accumulation would unite groups of phyla, and would surely lead to a scenario highly incompatible with clock models suggesting deep origins of clades (as they would all be more fossilisable).
Apologies but we don’t completely understand this point as ‘synapomorphy accumulation would unite groups of phyla’ is a little ambiguous. Of course, this is generally true, but our results suggest there was little opportunity to accumulate identifiable synapomorphies linking pairs, triplets or quartets of our 5 spiralian phyla.
In addition, clock results suggest rather long periods of time leading to the phyla, which would imply that there would have to be extremely slow rates of molecular evolution to yield the short early branches here. Also, it might be worth referring to papers compatible with this view, such as Wernström, J.V. et al., EvoDevo 13, 17 (2022). https://doi.org/10.1186/s13227-022-00202-8 or some of the palaeo literature, such as Budd and Jackson 2016, Phil Trans.
The referee refers to clock results suggesting a (deep) Ediacaran origin of Lophotrochozoa/Spiralia. We interpret the spiralian radiation itself as rapid but, in the absence of a clock analysis, we cannot comment on when it took place.
Reviewer #2 (Recommendations for the authors):
(My not very) Major points - as I feel this is an excellent paper.
(1) The coalescent-based summary tree analyses warrant expansion. The recovery of flatworms within a paraphyletic jaw-bearing animal clade in both summary trees is a striking result attributed to long-branch attraction, but this interpretation would be strengthened by examining whether pruning or downweighting the longest-branching taxa within those groups affects the outcome, or by reporting per-node quartet scores more fully. This would make the reticulation-aware results more directly informative and would bring this section into better balance with the detailed likelihood-based analyses.
We thank the reviewer for the suggestion of the expanded analyses. We have now done these, and they yielded essentially the same results as the unpruned analyses. Additionally, while not discussed, we ran the Astral analyses on the subset of gene-trees where all groups of interest (spiralian phyla and superphyletic Ecdysozoa, Deuterostomia, etc.) were monophyletic and found no changes to interphylum quartet scores beyond those due to enforced (super)phylum monophyly, with Platyhelminths still recovered within Gnathifera.
We have expanded our description of the results slightly as well as our discussion. Location of the tables with detailed quartet scores and local posterior probabilities has been added to Fig. S1’s legend.
(2) It would strengthen the paper to include at least a brief analysis or explicit discussion of whether any currently available models accounting for non-stationary or across-lineage compositional heterogeneity show any change in the pattern of support, even if only tested on a subset of topologies. A null result here would itself be informative and would make the conclusions more robust to the concern that unexamined model classes might behave differently.
We thank the reviewer for the suggestion, but this represents a considerable amount of new work and we think it falls outside the scope of the present work. We have, as suggested, included this as a discussion point.
(3) The authors note that topologies grouping flatworms with ribbon worms appear among the higher-scoring arrangements even under model misspecification in simulations. It would be helpful to comment explicitly on whether the apparent signal for this grouping should therefore be regarded with particular scepticism, or whether it survives artefact correction in any of the analyses, as this is a grouping that has appeared repeatedly in the literature and readers will want guidance on how to interpret it.
We do state that the nemertean+platyhelminth grouping seems likely to be at the least emphasised by an artefact (as the referee points out it is common to the higher scoring trees in the star tree simulations). We state that this suggests “…that this grouping derives some support from systematic errors.” We now return briefly to this in the discussion.
Writing and presentation
(1) The abstract states that rooting Spiralia on the flatworm branch "is a long-branch artefact" - this is slightly stronger than the language used in the body of the paper, where the authors correctly write that this preference is "at least enhanced by" the artefact. The abstract phrasing should be softened to reflect the more nuanced conclusion in the text.
Good point. Done.
(2) A brief signposting sentence near the start of the Results, setting out the overall analytical logic before the individual sections begin, would help orient readers. The strategy - score all topologies, test robustness to model choice and taxon sampling, then use simulation to identify artefactual signals - is clear in retrospect but would benefit from being made explicit upfront.
We have taken this suggestion on board. The summary seemed in the end better placed as the final part of the introduction.
(3) Figure 3 is complex and would be easier to interpret with a brief explanatory note in the legend clarifying what a wide versus narrow range of log-likelihood scores across topologies means in practical terms for statistical resolution between trees.
Added sentence to legend.
Minor Corrections:
(1) The Figure 2 legend contains a typographical error: "shorter than the short, disputed deuterostome branch" should read "shorter than."
Done
(2) At least one reference appears to carry a future publication year (Ishii et al., 2026) and should be verified for accuracy before final submission.
This reference is correct per the journal’s website. We did find Google Scholar to list it as being from 2025.
Reviewer #3 (Recommendations for the authors):
(1) Abstract/SI definitions of Spiralia/Lophotrochozoa
While I don't have strong feelings about this, if Spiralia is being used as an apomorphy-based name, then it still might be equivalent to Lophotrochozoa, as spiral cleavage in Gnathostoniula jenneri was illustrated by Riedl (1969). Although no other studies have replicated this observation, this should at least be mentioned.
Sorry this reference to gnathostomulid spiral cleavage was included in a longer version of the discussion of nomenclature. This was first reduced in length (which was when the mention of gnathostomulid spiral cleavage was dropped) then finally moved to the supplementary material. We have now re-included mention of this in the discussion in supplementary info.
The SI text suggests that the name Lophotrochozoa, as used in its original form by Halanych et al. (1995), was a node-based definition, and that this name is for the sister group of Ecdysozoa. However, in that paper, the name is actually defined as "as the last common ancestor of the three traditional lophophorate taxa, the molluscs, and the annelids, and all of the descendants of that common ancestor". This definition would exclude Gnathifera, and depending on the internal relationships of the non-Gnathiferan phyla, may be equivalent (or not) to the usage of the name Spiralia adopted in the present paper. The perils of mixing node and apomorphy-based definitions of clades are clear, and the situation is less straightforward than the paper suggests, and (somewhat unhelpfully given the subject of the paper) may only become clearer if the relationships of non-ecdysozoan protostomes are resolved.
We believe that the community universally understood the definition of Lophotrochozoa following the 1997 paper (by the authors who also provided the original 1995 definition). This 1997 definition included both chaetognaths and rotifers as examples of the Gnathifera. The Spiralia, in contrast, began life not even as a name for a clade but a description of a character shared by some apparently unrelated taxa – similar to a grouping of ‘carnivores’. The introduction of a new name was, we suggest, unhelpful. We hope that by defining our terms up front the meaning in the current paper is clear.
(2) Introduction
Line 76. Some references needed regarding claims that there was a polymeric brachiopod ancestor, e.g. Gutman (1978), Temereva and Malakhov (2011), Guo et al. (2023). Likewise for the chaetae of brachiopods, annelids and molluscs, e.g. Schiemann (2017), as it's key to trace where these ideas originated.
Added
Figure 1. This is a nice illustration of the uncertainty in the relationships of these groups. However, I kept checking which thumbnail image was which for nemerteans and annelids. A minor suggestion, but perhaps a polychaete instead for the annelid?
We have replaced the rather poor image of an earthworm with a polychaete and also now include labels. We hope the improved images are more helpful. Good point.
(3) Results
Branch length comparison. I understand why the deuterostome stem was chosen as the branch for comparison from the point of view of phylogenetic uncertainty. However, what about the branch leading to ecdysozoa or the branch subtending lophotrochozoan and/or gnathifera? Given that the short internodes are used as an argument underpinning uncertain relationships, can we be sure that Gnathifera is not nested within the group of interest, especially given that Gnathifera contains many long-branched taxa and the root may be misplaced within the group?
We have added the Lophotrochozoa and Ecdysozoa median lengths to our plots and now discuss both the lophotrochozoan branch in our results.
Line 249. Given that Spiralia is the group of interest, why were the Gnathiferans also chosen at random?
The point of the experiment was to see the effect of taxon sampling on the consistency of the resulting topology. Random sampling across the tree seems helpful in this context. We chose Gnathifera as one group to sample from as this ensured they would be present in all trees. This seems appropriate as they are the sister group of the clade of interest and as such their inclusion reflects a choice a typical investigator might make when choosing which species to include. Additionally, as noted in the reviewer’s earlier comment, Gnathifera includes many long-branched taxa and we wanted to ensure our root-placement results were robust to this aspect of taxon sampling.
(4) Discussion
Line 448. Our current understanding of the early spiralian fossil record is quite consistent with the main results of this paper. For example, there are very few claims for fossils that sit on the short branch leading to Spiralia (or Lophotrochozoa as defined here) that this paper discusses. Many of the key fossils that inform on the characters discussed in the introduction that have unusual character combinations have an apomorphy of one of the phyla discussed, and so are resolved as members of the stem lineages of particular phyla.
This is what you would expect with long phylum stem lineages (line 148) and a short spiralia stem lineage. For example, the mollusc Wiwaxia has chaetae, but a mollusc like radula (Smith 2012), the conchiferan mollusc Pelagiella has chaetae and a coiled shell (Thomas et al. 2020). The only fossil groups that are routinely discussed as belonging to the stem lineage of more than one phylum are the tommotiids, which have chaetae, segmentation and a complex mineralised skeleton (but not shells in the brachiopod/mollusc sense, see Guo et al 2023) but they sit on the lophophorate stem lineage, a synapomorphy rich group the monophyly of which the present paper endorses (e.g. line 435). The fossil record is consistent with the scenario presented in line 442, e.g. convergent loss or reduction of chaetae and segmentation and convergent evolution of shells in molluscs and brachiopods.
We accept these points (though are clearly not experts on these fossils). We have (slightly tentatively given our lack of expertise) expanded our discussion to include these fossil taxa with their combinations of characters.
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eLife Assessment
This important study probes the long-standing failure to resolve evolutionary relationships between the classical "spiralian" taxa - i.e., annelids, molluscs, brachiopods, platyhelminths and nemerteans - and provides convincing evidence that the branches leading to them are so short as to be unreliable guides to their relationships. This, in turn, has wide-ranging implications for our understanding of animal body plan evolution and the interpretation of early animal fossils.
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Reviewer #1 (Public review):
Summary:
This interesting paper probes the problematic relationships between the classical "spiralian" taxa, i.e., annelids, molluscs, brachiopods, platyhelminths and nemerteans, and shows that the branches leading to them are so short as to be unreliable guides to their relationships. This, in turn, has important implications for how we view the origin of the animal phyla.
Strengths:
A very careful analysis of a famous old problem with quite significant results. The results seem to be robust and support their conclusions.
It often passes uncommented that many different trees are published about animal relationships, yet some parts of the tree seem extremely difficult to resolve; the spiralians are perhaps the most difficult case. More recently, problems about sponges or ctenophores as sister groups to the …
Reviewer #1 (Public review):
Summary:
This interesting paper probes the problematic relationships between the classical "spiralian" taxa, i.e., annelids, molluscs, brachiopods, platyhelminths and nemerteans, and shows that the branches leading to them are so short as to be unreliable guides to their relationships. This, in turn, has important implications for how we view the origin of the animal phyla.
Strengths:
A very careful analysis of a famous old problem with quite significant results. The results seem to be robust and support their conclusions.
It often passes uncommented that many different trees are published about animal relationships, yet some parts of the tree seem extremely difficult to resolve; the spiralians are perhaps the most difficult case. More recently, problems about sponges or ctenophores as sister groups to the rest of the animals have alerted us to major areas of uncertainty in large-scale phylogenetic reconstruction; this paper is a welcome reminder that other, perhaps even harder, problems exist which may be difficult to ever resolve with the (molecular) data we have.
Weaknesses:
The paper could have perhaps drawn out some of the implications of its results in a clearer manner.
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Reviewer #2 (Public review):
Summary:
The relationships among the phyla making up Spiralia - a major clade of animals including molluscs, annelids, flatworms, nemerteans and brachiopods - have been challenging from a phylogenomic perspective despite decades of molecular phylogenetic effort. Every topology uniting subsets of these phyla has been recovered with apparent support in at least one study, yet no consensus has emerged even from large-scale genomic datasets. Serra Silva and Telford set out to determine whether this instability reflects a genuine biological signal being obscured by analytical limitations, or whether it reflects a rapid, near-simultaneous origin of these phyla that has left behind in modern genomes far too little phylogenetic information to resolve. They focused deliberately on five phyla, reducing the problem to …
Reviewer #2 (Public review):
Summary:
The relationships among the phyla making up Spiralia - a major clade of animals including molluscs, annelids, flatworms, nemerteans and brachiopods - have been challenging from a phylogenomic perspective despite decades of molecular phylogenetic effort. Every topology uniting subsets of these phyla has been recovered with apparent support in at least one study, yet no consensus has emerged even from large-scale genomic datasets. Serra Silva and Telford set out to determine whether this instability reflects a genuine biological signal being obscured by analytical limitations, or whether it reflects a rapid, near-simultaneous origin of these phyla that has left behind in modern genomes far too little phylogenetic information to resolve. They focused deliberately on five phyla, reducing the problem to a tractable set of 15 unrooted and 105 rooted topologies, and applied a suite of complementary approaches across two independent datasets and multiple substitution models to test whether any topology is significantly preferred over alternatives.
Strengths:
(1) The conceptual framing of the problem is excellent, and the study makes a convincing case across several lines of evidence. By enumerating all possible topologies and demonstrating empirically that every one of the 15 unrooted arrangements has been recovered as the preferred solution in at least one published study, the authors make a strong argument about the state of the field. The use of two entirely independent datasets as a consistency check is great, and convergence between them, where it occur,s substantially strengthens confidence in the conclusions.
(2) It is my view that the simulation framework is a particular strength. Generating data on a fully unresolved star tree and scoring those data under both correctly-specified and misspecified substitution models provides convincing evidence that the strong preference for rooting Spiralia on the flatworm branch is, at least partly, an analytical artefact driven by the exceptionally long branch in combination with compositional heterogeneity across sites. This is an important methodological demonstration with implications beyond spiralian phylogenetics, as the same issue is likely to affect other deep, long-branched lineages in the animal tree of life.
(3) The randomised taxon-jackknifing approach is a very nice addition here. The demonstration that preferred topologies shift depending on which species happen to be sampled (even within the same phylum) is a convincing indicator of weak signal, and provides a practical caution for future studies that may report strong support for a particular spiralian arrangement based on a fixed taxon sample.
(4) The branch-length analyses, benchmarking internal interphylum branches against the already disputed and extremely short branch uniting deuterostomes (work also by this group), are well-conceived and solid.
(5) I think it is worth highlighting the notable intellectual honesty throughout the paper: the authors do not overstate their results, correctly acknowledging that while the unrooted topology grouping molluscs with brachiopods and flatworms with nemerteans emerges most consistently, this preference is not statistically significant under more adequate substitution models and may itself carry some artefactual component.
Weaknesses:
(1) The restriction to five phyla is the most significant limitation, as the authors acknowledge this and give a clear computational justification, but readers should be aware that the paper's convincing conclusions apply specifically to the five focal phyla and the evidence remains incomplete with respect to spiralian phylogeny as a whole.
(2) The treatment of substitution model adequacy, while commendably thorough for site-heterogeneous models, is necessarily bounded. The authors note that models accounting for non-stationarity, across-lineage compositional heterogeneity, or mixtures of tree histories might yield different results, and that even the most sophisticated currently available approaches have not produced consistent spiralian topologies across studies. This is not a criticism of what has been done here - the analytical scope is reasonable and well-implemented - but it means the paper cannot be read as a definitive demonstration that no model will ever resolve these relationships. The distinction between a true hard polytomy and a radiation that is effectively unresolvable given current data and methods could be drawn more sharply in the discussion.
(3) The reticulation-aware coalescent analyses are presented somewhat briefly relative to the likelihood-based topology scoring. The finding that flatworms are recovered within a paraphyletic jaw-bearing animal clade in both summary trees - interpreted as long-branch attraction - is striking, and its implications for gene-tree-based approaches to spiralian rooting deserve more discussion than they currently receive.
(4) The central conclusions - that interphylum branches in Spiralia are extraordinarily short, that topological preferences are strongly model-dependent and taxon-sampling-sensitive, and that an ancient rapid radiation is the most parsimonious explanation - are convincingly supported by the evidence presented. The identification of flatworm long-branch attraction as an important confounding factor in rooting analyses is itself an important and well-demonstrated result.
Conclusion:
This paper clearly makes an important contribution to the ongoing debate about spiralian relationships and, more broadly, to methodological discussions about how to handle anciently diversified clades where phylogenetic signal is genuinely limited. The exhaustive topology-scoring framework combined with taxon-jackknifing and simulation under unresolved trees is a valuable methodological template that could usefully be applied to other notoriously difficult nodes in the animal tree. I thoroughly enjoyed the discussion of the implications of these findings for interpreting Cambrian fossils and the evolutionary history of shells, segmentation, larval types and other characters - it is both thoughtful and thought-provoking and will be of broad interest well beyond the phylogenomics and zoology communities. From a very practical perspective, the data and scripts provided make the work useful to researchers wishing to apply similar approaches to other groups.
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Reviewer #3 (Public review):
Summary:
This paper addresses the controversial internal relationships within the Spiralia, a major clade of invertebrate animals including molluscs, annelids, brachiopods and flatworms.
Strengths:
Performs a range of empirical analyses and simulations that address the core question. Although a favoured unrooted topology finds some support, this is not strongly endorsed in the paper.
Weaknesses:
(1) Only considers a subset of relevant phyla (e.g. gastrotrichs are relevant to the phylogenetic position of Platyhelminthes), although how this would change the scale of the analyses (i.e. number of topologies) is addressed in the paper.
(2) Discussion of Spiralia evolution and broader context, particularly the relevance for the fossil record. Line 448: our current understanding of the early spiralian fossil record …
Reviewer #3 (Public review):
Summary:
This paper addresses the controversial internal relationships within the Spiralia, a major clade of invertebrate animals including molluscs, annelids, brachiopods and flatworms.
Strengths:
Performs a range of empirical analyses and simulations that address the core question. Although a favoured unrooted topology finds some support, this is not strongly endorsed in the paper.
Weaknesses:
(1) Only considers a subset of relevant phyla (e.g. gastrotrichs are relevant to the phylogenetic position of Platyhelminthes), although how this would change the scale of the analyses (i.e. number of topologies) is addressed in the paper.
(2) Discussion of Spiralia evolution and broader context, particularly the relevance for the fossil record. Line 448: our current understanding of the early spiralian fossil record is quite consistent with the main results of this paper. For example, there are very few claims for fossils that sit on the short branch leading to Spiralia (or Lophotrochozoa as defined here) that this paper discusses. Many of the key fossils that inform on the characters discussed in the introduction, which have unusual character combinations, have an apomorphy of one of the phyla discussed, and so are resolved as members of the stem lineages of particular phyla.
(3) This is what you would expect with long phylum stem lineages (line 148) and a short spiralia stem lineage. For example, the mollusc Wiwaxia has chaetae, but a mollusc like Radula (Smith 2012), the conchiferan mollusc Pelagiella has chaetae and a coiled shell (Thomas et al. 2020). The only fossil groups that are routinely discussed as belonging to the stem lineage of more than one phylum are the tommotiids, which have chaetae, segmentation and a complex mineralised skeleton (but not shells in the brachiopod/mollusc sense, see Guo et al 2023) but they sit on the lophophorate stem lineage, a synapomorphy rich group the monophyly of which the present paper endorses (e.g. line 435). The fossil record is consistent with the scenario presented in line 442, e.g. convergent loss or reduction of chaetae and segmentation and convergent evolution of shells in molluscs and brachiopods.
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