Reviewer 1: Dr. Eugene Koonin

Panchin et al. propose the SCANDAL hypothesis under which certain groups of secondarily simplified animals, in particular, Myxosporea, evolved from transmissible cancers. The hypothesis is both obvious enough - after all, the transmissible tumors are a step ahead of metastases on the path of tumor autonomization, and it is natural to speculate on the next step - and scandalous enough which, I suppose, is a good feature for a hypothesis. The discussion in the article is well informed, with respect to both transmissible tumors and animal evolution, and interesting. The authors make a laudable attempt to render the hypothesis falsifiable by postulating that any serious candidates for the origin from trnasmissible tumors should have lost a substantial fraction of tumor suppressors and apoptosis effectors. This is a (overly?) stringent criterion because there are many pathways to cancer, not necessarily through currently known “cancer genes”, and many ways to impair apoptosis as well. So, in a sense, this amounts to searching under the streetlight, but I think the approach is appropriate as a strong selection criterion for dismissing SCANDAL candidate. Indeed, by applying it, the authors discard 3 of the 4 initially suggested groups, and zero in on Myxosporea that have indeed lost quite a few of the genes of interest. As the authors admit, this cannot be really considered “support” for the hypothesis, only observations that appear compatible with it. Again, as the authors rightly note, the loss of these genes seems to be part of the general trend of reductive evolution, with no evidence that it was selective with respect to the cancer-related genes. My main question is very simple: do Cnidaria have cancer? If so, even if transmissibility has not been demonstrated, then, the origin of Myxosporea from a tumor would appear a distinct possibility. If not, the entire scenario is entirely speculative. On the whole, I tend to think that the hypothesis is wrong, and there is no SCANDAL in the animal kingdom. However, the possibility that there is definitely merits discussion, so the article will be of interest to many and could stimulate new insight and actual new research.

Authors response

We thank Dr. Koonin for his comment on our hypothesis. We decided that the answer to the question “do Cnidaria have cancer?” should be better presented in the main text of our article so we added the following paragraph:

“Calicoblastic neoplasms have been found in corals and are characterized by rapid growth, loss of differentiation, loss of tissue architecture, proliferation of gastro-vascular canals and fitness reduction [35, 36]. Tumors were also recently identified in Hydra [37]. A transriptome analysis suggested that some of the misregulated genes of these tumors are homologous to mammalian tumor-related genes, including those involved in apoptosis [37] The cells in these tumors were invasive and could be viewed as metastatic”.

Reviewer 2: Dr. Mikhail Gelfand

The hypothesis presented in the paper is likely wrong, but clever and interesting. I commend the authors for citing not only supporting evidence (loss of many cancer-relate domains), but evidence against it (retention of some domains and same rates of loss in cancer-related and control domains).

The only technical point I can make relates to identification of domains via analysis of ORFs (in the absence of published annotation). Could it be that the exon length in some species is so small that domains might have been missed by the applied procedure? A control could be systematic search for some other domains using the same procedure. Similarly, to be on the safe side, it might be a good idea to check for possible underannotation by searching for the missing domains in Myxosporea genomes directly, not relying on annotation. The authors mention hamster reticulum cell sarcoma as an example of transmissible cancer. This cancer is not mentioned in a number of recent reviews (e.g Ujvari, Papenfuss & Belov, Bioessays, 2016 and Riquet, Simon & Bierne, Evol. Appl., 2016). The most likely explanation is that the authors of these reviews have missed old publications, in particular, the 1964 one cited by the authors. However, there seems to be some conflicting evidence: while a 1965 paper (Banfield, Woke, Mackay & Cooper, Science, 1965) states that mosquitoes transmit cancer cells and not any other oncogenic agent, a 1973 paper suggests that reticulum cell sarcoma of Syrian hamsters is caused by SV40 virus (Diamandopoulos, J. Natl. Cancer Inst., 1973). Could that be a different type of this cancer, or could the authors of the 60’s papers miss this possibility? In interesting issue, not directly covered by the authors, is what genes should have evolved (or re-evolved) to regain multicellularity of Myxosporea, if the hypothesis of their cancer, unicellular origin is true.

The sentence “multicellularity evolved independently at least 46 times in eukaryotes” clearly contains a misprint. The grammar needs to be checked, e.g. some commas and articles are clearly redundant.

Authors response

We thank Dr. Gelfand for his suggestions and questions.

The five Myxozoa species used in our analysis had different types of high-throughput sequencing data available. For three species (Kudoa iwatai, Sphaeromyxa zaharoni and Enteromyxum leei) we had genomic data, for one (Myxobolus cerebralis) we used transcriptome data and proteome data was available for the final species (Thelohanellus kitauei). We obtained similar results of domain loss for all five species. Thus, it is unlikely that some domains were lost due to small exon length. In addition, selected cases of domain loss were additionally tested by BLAST searches seeded with human orthologs. No disagreement with HMMER search of PFAM domains was found.

Ostrander et al. recently reviewed the case of the Syrian hamster sarcoma [7]. It appears that several research groups studied different cases of hamster tumors. Apparently, the SV40 virus can cause sarcomas, but it is not clear if a viral agent caused the original reticulum cell sarcoma studied in the article we cite. We added a comment that the viral origin of such tumors is an alternative possibility to our introduction.

The re-evolution of genes to regain multicellularity is an interesting question. However, it is not clear how to search for such genes. It is unlikely that such re-evolved genes share sequence similarity to those that were required for multicellularity before, if the original genes were lost, even if they provide the same functions. It is also worth mentioning that the multicellularity of Myxosporea is quite different from that of other Cnidaria.

We thank the reviewer for noticing an error. In a 2007 review Grosberg et al. write that “Multicellular organisms independently originated at least 25 times from unicellular ancestors” [49]. In a 2013 review Parfrey et al. state that “Multicellularity has arisen more than 25 times across the eukaryotic tree of life and in all of the major clades” [50]. We corrected the text.

Reviewer 3 Dr. Gregory M Woods

Initial submission

The hypothesis is that “Some simplified relatives of complex metazoans can have a tumor origin.” This is intriguing, and the authors provide some supporting evidence. The authors propose that “Relatives of more complex metazoans have genomic alterations typical for cancer progression (such as deletions of universal apoptosis genes).” However, if this was the case, some control would be required to prevent continuous “cancerous” growth. The deletion of apoptotic genes appears to be the authors’ major support of the “tumor” origin theory. The manuscript proposes an original hypothesis that initially appears implausible, but the authors propose some logical steps from cancer cell to a species. But the evidence is not compelling and selective (apoptotic genes).

This is an original hypothesis, which requires an open mind to seriously consider. Major recommendations Page 10 line 11 “Thus, we can put forward the hypothetical steps of Myxosporea evolution. First, a parasitic polypodium-like organism produced a tumor in a fish host. Some genes related to apoptosis and cell-cycle control were lost. This tumor acquired transmissibility as in known mammalian and molluscan transmissible cancers. Two different evolutionary trends followed and produced the modern Myxosporea. One trend was caused by the cancerous origin of Myxosporea species: multiple genes involved in apoptosis, cell-cycle suppression and associated pathways were abruptly lost. A different trend was directed towards a de novo formation of multicellularity resulting in the currently observed strange organisms with three cells stages and bizarre cell aggregates like syncitia with whole cells inside other cells.” Why was it necessary for the tumor to be polypodium like? How does this differ from speciation or evolution? Was this tumor polypodium in origin, or did it transform a host cell? How did the tumor acquire transmissibility? If it was a “mutated” parasite, then it could be transmitted. But if it was a transformed somatic, or even stem cells as the authors intimate, then a mechanism of transfer is required. Further, if it was a somatic cell, how did it acquire genes to allow it to survive outside the host, in parasitic form? Genes controlling apoptosis are essential for the ‘sculpting’ of multicellular organisms, especially those with defined organs. How can Myxosporea survive without apoptotic genes? Clearly, Myxosporea exist, so there must be an alternative mechanisms. Evidence was provided for the loss of apoptotic related genes and p53, but what were the associated pathways. What were the mechanisms of de novo formation of multicellularity. A reference was quoted but mechanism not explained. It wasn’t clear whether the tumor cell was initially a parasite or a somatic cell. If the former, how is this different from ‘evolution’ and if the latter, how was the stem cell sculpted into a multi-cellular organism. Minor recommendations Page 2, line 18 – “Given the ability of some tumors to survive for thousands of years [5]” – One tumor, CTVT, has existed for thousands of years. Page 2 line 19 their progenitors as in the case of the canine transmissible venereal tumor [10], it is possible for them to evolve into a new species. How can this be possible? Big jump from complex mammals to simple metazoans.

Authors response (initial submission)

We thank Dr. Woods for his comment and understand his concerns. We would like to answer his questions in a point-by-point manner.

1. Why was it necessary for the tumor to be Polypodium like?

We do not state that the tumor needs to be Polypodium like. We merely state that Polypodium is the closest phylogenetic group relative of Myxozoa among presently known Сnidaria. If Myxosporea evolved as a SCANDAL, then their closest relatives give us our best guess about how their ancestors looked like.

2. How does this differ from speciation or evolution?

The difference is that there was (as we hypothesize) a catastrophic simplification of Myxosporean ancestors through a tumor stage, followed by a de novo acquisition of multicellularity. This would be a very unusual process of evolution and speciation.

3. Was this tumor Polypodium in origin, or did it transform a host cell?

We hypothesize that it was a tumor of Polypodium that spread to the host. Myxosporeans are genetically related to other Cnidaria, not their fish or annelid hosts. We clarified this in the text.

4. How did the tumor acquire transmissibility?

We do not know the exact mechanism. However, we know that some tumors acquire transmissibility through some mechanism.

5. Further, if it was a somatic cell, how did it acquire genes to allow it to survive outside the host, in parasitic form?

We believe this point is similar to point 3.

6. e6. Genes controlling apoptosis are essential for the ‘sculpting’ of multicellular organisms, especially those with defined organs. How can Myxosporea survive without apoptotic genes?

They do not have defined organs and their multicellularity is different from that of most Metazoa.

7. What were the mechanisms of de novo formation of multicellularity

We do not know.

8. It wasn’t clear whether the tumor cell was initially a parasite or a somatic cell

Our hypothesis is that a Cnidaria somatic cell became first a tumor and then a transmissible parasite. Actually, there could be two different scenarios: 1) a free-living Cnidarian acquired a transmissible tumor that later infected a different species (fish or annelid). 2) a parasitic Cnidarian acquired a transmissible tumor that later adopted transmission between host species. In our example scenario, we preferred the second possibility because the fish parasite Polypodium is the closest known phylogenetic relative of Myxozoa. Myxosporea and Malacosporea are fish parasites.

9. How can this be possible? Big jump from complex mammals to simple metazoans.

We know that multicellularity has evolved independently more than a few times on the tree of life. Therefore, given the possibility for a line of transmissible cancer cells to exist for thousands of years, it is possible that they can exist longer, long enough to evolve multicellularity. We do not claim that this is a probable or likely scenario, but we do believe that it is possible. If complex mammals can acquire transmissible tumors, why not Cnidaria?

We thank the reviewer for the suggested minor correction about the CTVT tumor sentence.

Reviewer 3 (revision 1): Gregory M woods

Although the SCANDAL hypothesis is unlikely, it is intriguing, and worth publishing as a hypothesis. The authors responses were minimal but generally adequate, with two minor exceptions (below). How does this differ from speciation or evolution? “The difference is that there was (as we hypothesize) a catastrophic simplification of Myxosporean ancestors through a tumor stage, followed by a de novo acquisition of multicellularity. This would be a very unusual process of evolution and speciation.” Agreed and understood, but the authors’ hypothesis is also a “very unusual process”. There was no mechanism mentioned for acquisition of multicellularity. It would have been beneficial to respond to the question following the above paragraph in the text. If I have misinterpreted, especially as evolution is frequently mentioned, others may. How did the tumor acquire transmissibility? We do not know the exact mechanism. However, we know that some tumors acquire transmissibility through some mechanism. But please provide potential examples rather than “some mechanisms”. Transmissibility is central to the overall hypothesis.

Author’s response

We thank the reviewer for his further comments about our hypothesis. We think that we were not correctly understood. The phrase “very unusual process” was referring to our hypothesis. The process we describe is evolution and it is speciation, but with an unusual detail: a multicellular organism gets a tumor, the tumor evolves and becomes transmissible, and the transmissible tumor evolves and becomes a new multicellular species. Examples of first two stages exist and we mention them in our introduction (the Tasmanian devil facial tumor disease, the canine transmissible venereal tumor, transmissible cancers of bivalve mollusks). Examples of single cell organisms evolving into multicellular species are also known. Here we refer to reviews [49, 50]. However, it is yet unknown if a sequence of all three stages has occurred in nature. Our comparative genomic analysis suggests that this could be true for Myxosporea evolution. However, additional studies are required to confirm or refute this hypothesis and to propose a more detailed mechanism of multicellularity acquisition in this case (if the hypothesis is true).