Among living vertebrates, there is broad consensus that living tetrapods consist of amphibians and amniotes. Crown clade Lissamphibia contains frogs (Anura), salamanders (Urodela) and caecilians (Gymnophiona); Amniota contains Sauropsida (reptiles including birds) and Synapsida (mammals). Within Lissamphibia, most studies place frogs and salamanders in a clade together to the exclusion of caecilians (see Pyron & Wiens 2011). Among fossils, there are a number of amphibian and amphibian-like taxa generally placed in Temnospondyli and Lepospondyli. In contrast to the tree of living tetrapods, affinities of these fossils to some or all of the three extant lissamphibian groups have proven to be much harder to resolve. For example, temnospondyls might be stem tetrapods and lissamphibians a derived group of lepospondyls; alternatively, temnospondyls might be closer to the clade of frogs and salamanders, and lepospondyls to caecilians (compare Laurin et al. 2019: fig. 1d vs. 1f). Here, in order to assess which of these and other mutually exclusive topologies is optimal, Laurin et al. (2019) extract phylogenetic information from developmental sequences, in particular ossification. Several major differences in ossification are known to distinguish vertebrate clades. For example, due to their short intrauterine development and need to climb from the reproductive tract into the pouch, marsupial mammals famously accelerate ossification of their facial skeleton and forelimb; in contrast to placentals, newborn marsupials can climb, smell & suck before they have much in the way of lungs, kidneys, or hindlimbs (Smith 2001). Divergences among living and fossil amphibian groups are likely pre-Triassic (San Mauro 2010; Pyron 2011), much older than a Jurassic split between marsupials and placentals (Tarver et al. 2016), and the quality of the fossil record generally decreases with ever-older divergences. Nonetheless, there are a number of well-preserved examples of "amphibian"-grade tetrapods representing distinct ontogenetic stages (Schoch 2003, 2004; Schoch and Witzmann 2009; Olori 2013; Werneburg 2018; among others), all amenable to analysis of ossification sequences. Putting together a phylogenetic dataset based on ossification sequences is not trivial; sequences are not static features apparent on individual specimens. Rather, one needs multiple specimens representing discrete developmental stages for each taxon to be compared, meaning that sequences are usually available for only a few characters. Laurin et al. (2019) have nonetheless put together the most exhaustive matrix of tetrapod sequences so far, with taxon coverage ranging from 62 genera for appendicular characters to 107 for one of their cranial datasets, each sampling between 4-8 characters (Laurin et al. 2019: table 1). The small number of characters means that simply applying an optimality criterion (such as parsimony) is unlikely to resolve most nodes; treespace is too flat to be able to offer optimal peaks up which a search algorithm might climb. However, Laurin et al. (2019) were able to test each of the main competing hypotheses, defined a priori as a branching topology, given their ossification sequence dataset and a likelihood optimality criterion. Their most consistent result comes from their cranial ossification sequences and supports their "LH", or lepospondyl hypothesis (Laurin et al. 2019: fig. 1d). That is, relative to extinct, "amphibian"-grade taxa, Lissamphibia is monophyletic and nested within lepospondyls. Compared to mammals and birds (including dinosaurs), crown amphibian branches of the Tree of Life are exceptionally old. Each lissamphibian clade likely had diverged during Permian times (Marjanovic & Laurin 2008) and the crown group itself may even date to the Carboniferous (Pyron 2011). In contrast to mammoths and moas, no ancient DNA or collagen sequences are going to be available from >300 million-year-old fossils like the lepospondyl *Hyloplesion* (Olori 2013), although recently published methods for incorporating genomic signal from extant taxa (Beck & Baillie 2018; Asher et al. 2019) into studies of fossils could also be applied to these ancient divergences among amphibian-grade tetrapods. Ossification sequences represent another important, additional source of data with which to test the conclusion of Laurin et al. (2019) that monophyletic Lissamphibians shared a common ancestor with lepospondyls, among other hypotheses. **References** Asher, R. J., Smith, M. R., Rankin, A., & Emry, R. J. (2019). Congruence, fossils and the evolutionary tree of rodents and lagomorphs. Royal Society Open Science, 6(7), 190387. doi: [ 10.1098/rsos.190387 ](https://dx.doi.org/ 10.1098/rsos.190387 ) Beck, R. M. D., & Baillie, C. (2018). Improvements in the fossil record may largely resolve current conflicts between morphological and molecular estimates of mammal phylogeny. Proceedings of the Royal Society B: Biological Sciences, 285(1893), 20181632. doi: [ 10.1098/rspb.2018.1632](https://dx.doi.org/ 10.1098/rspb.2018.1632) Laurin, M., Lapauze, O., & Marjanović, D. (2019). What do ossification sequences tell us about the origin of extant amphibians? BioRxiv, 352609, ver. 4 peer-reviewed by PCI Paleo. doi: [ 10.1101/352609](https://dx.doi.org/ 10.1101/352609) Marjanović, D., & Laurin, M. (2008). Assessing confidence intervals for stratigraphic ranges of higher taxa: the case of Lissamphibia. Acta Palaeontologica Polonica, 53(3), 413–432. doi: [ 10.4202/app.2008.0305](https://dx.doi.org/ 10.4202/app.2008.0305) Olori, J. C. (2013). Ontogenetic sequence reconstruction and sequence polymorphism in extinct taxa: an example using early tetrapods (Tetrapoda: Lepospondyli). Paleobiology, 39(3), 400–428. doi: [ 10.1666/12031](https://dx.doi.org/ 10.1666/12031) Pyron, R. A. (2011). Divergence time estimation using fossils as terminal taxa and the origins of Lissamphibia. Systematic Biology, 60(4), 466–481. doi: [ 10.1093/sysbio/syr047](https://dx.doi.org/ 10.1093/sysbio/syr047) Pyron, R. A., & Wiens, J. J. (2011). A large-scale phylogeny of Amphibia including over 2800 species, and a revised classification of extant frogs, salamanders, and caecilians. Molecular Phylogenetics and Evolution, 61(2), 543–583. doi: [ 10.1016/j.ympev.2011.06.012](https://dx.doi.org/ 10.1016/j.ympev.2011.06.012) San Mauro, D. (2010). A multilocus timescale for the origin of extant amphibians. Molecular Phylogenetics and Evolution, 56(2), 554–561. doi: [ 10.1016/j.ympev.2010.04.019](https://dx.doi.org/ 10.1016/j.ympev.2010.04.019) Schoch, R. R. (2003). Early larval ontogeny of the Permo-Carboniferous temnospondyl *Sclerocephalus*. Palaeontology, 46(5), 1055–1072. doi: [ 10.1111/1475-4983.00333](https://dx.doi.org/ 10.1111/1475-4983.00333) Schoch, R. R. (2004). Skeleton formation in the Branchiosauridae: a case study in comparing ontogenetic trajectories. Journal of Vertebrate Paleontology, 24(2), 309–319. doi: [ 10.1671/1950](https://dx.doi.org/ 10.1671/1950) Schoch, R. R., & Witzmann, F. (2009). Osteology and relationships of the temnospondyl genus *Sclerocephalus*. Zoological Journal of the Linnean Society, 157(1), 135–168. doi: [ 10.1111/j.1096-3642.2009.00535.x](https://dx.doi.org/ 10.1111/j.1096-3642.2009.00535.x) Smith, K. K. (2001). Heterochrony revisited: the evolution of developmental sequences. Biological Journal of the Linnean Society, 73(2), 169–186. doi: [ 10.1111/j.1095-8312.2001.tb01355.x](https://dx.doi.org/ 10.1111/j.1095-8312.2001.tb01355.x) Tarver, J. E., dos Reis, M., Mirarab, S., Moran, R. J., Parker, S., O’Reilly, J. E., & Pisani, D. (2016). The interrelationships of placental mammals and the limits of phylogenetic inference. Genome Biology and Evolution, 8(2), 330–344. doi: [ 10.1093/gbe/evv261](https://dx.doi.org/ 10.1093/gbe/evv261) Werneburg, R. (2018). Earliest “nursery ground” of temnospondyl amphibians in the Permian. Semana, 32, 3–42.
I agree with the reviewer that this manuscript is just about ready for publication. I've made a number of minor comments for the authors to consider, below, so am obliged to tick "revision". However these are all minor and the authors can incorporate as they see fit.
line 14: I'd slightly edit the first sentence. "Controversial" is a value judgement; I'd delete this term. The authors say as much in the end of this sentence regarding current lack of consensus. You might also add some text to make clear that the lack of consensus is about lissamphibian affinities among fossil groups, not (for example) that they are tetrapods or are the sister taxon to extant amniotes.
line 37: There's only one phylogeny (at least of vertebrate high level taxa) and it's neither "molecular" nor "paleontological". Rather, we use these kinds of data (among others) to reconstruct what it is. So here & throughout reserve adjectives like "molecular" to describe data, not phylogenies (at least when you're talking about species trees and not gene trees). The authors already use this style nicely in (for example) lines 58, 82, 93, 97.
line 44: Note that Gill (1872 Smithsonian Misc Collec, p. xliii) shows a very modern-looking tree with well-corroborated clades like gnathosomtes, cyclostomes, bony fish, actinopts, sarcopts including lungfish & coelacanth, and tetrapods with "reptiles" originating out of "batrachians" (see also discussion in Asher & Müller 2012 chap 1, p.2 in From Clone to Bone CUP)
lines 111-112: This sentence is a bit long. I'd recommend "...extant amphibians. Recently, Danto et al. (2019) ..."
line 134: We may need some guidance from our PCI colleagues regarding "supplementary material". At present, this is mentioned in the main text but without a URL or other precise description of exactly where this is (or will be) available. Reference to the URL should eventually be added either as an appendix or directly in the text whenever "supplementary data" or "supplement" is mentioned (e.g., line 270). Relatedly, ensure that the wording for these data is the same throughout, as opposed to writing "supplementary material" on line 134 and "supplement" on line 270.
lines 182-183: The comment about lungfish "seem mostly impossible to homologize" is ambiguous. Ideally the authors might add a bit more justification or background to this statement. I'd recommend adding at least some references to guide readers to previous efforts at recognizing cranial homologies in these groups. Also please respond to the comment from R1 regarding line 133 in the first version: "133- this is incorrect. Firstly, Schoch 2006 used the actinopt Amia with fairly few homology problems. Secondly, some part of the development of Eusthenopteran were published (Cote, 2002; Schultze 1984), though admittedly little about cranial development. It would provide some data about postcranial though."
line 217: "see below" regarding missing data might also pertain to the text above
line 237: the sentence here can end with "...events in ontogeny" without the "would". The following sentence is confusing; it has too many clauses (between commas) and two occurrences of "because". Please rewrite.
line 241: "simple" regarding your formula is a value judgment and should be deleted.
lines 260-261: change to "...only with sequences standardized by position" (or otherwise simplify so that "sequence" isn't repeated). Also "data" as a plural should be modified by "few" rather than "little".
line 292-93: change "maximal" to "maximum". The fact that the analysis took so long is presumably because with few characters the treespace is relatively flat and the algorithm gets bogged down on many local optima. I've found the recent builds of PAUP (including 4.0a165 used here) are faster than 4.0b10 from a few years ago, but (unless you explicitly limit PAUP, e.g., time or iterations per replicate) it'll get stuck among the huge number of possible topologies in a flat-treespace with few characters. Default in TNT is faster; you can limit the tree buffer in advance via "hold" and (in my experience) it will more quickly escape local optima, only filling up the tree buffer upon branch swapping & after finding many local optima. There's no need to redo analyses as far as I can see but for future reference, the authors might consider using TNT: http://phylo.wikidot.com/tntwiki http://phylobotanist.blogspot.com/2015/03/parsimony-analysis-in-tnt-using-command.html https://groups.google.com/forum/#!forum/tnt-tree-analysis-using-new-technology
line 329: It's not quite clear to me why "branch lenghts... set to the same length" is an expectation of punctuated equilibria. The latter is (despite occasional Gouldian hyperbole) is just an application of peripatric (= "allopatric" in Eldredge & Gould 1972) speciation to the fossil record & the consequent expectation that small populations will tend not to leave behind a fossil record. "Stasis is data", as the saying goes, & is essentially an indication of population size. Perhaps another sentence or two explaining how punk-eek leads to particular expectations regarding branch lengths would be helpful.
line 350: "mentioned" is more appropriate than "evoked"
line 359: change to "...drawbacks that led us not to use them".
line 361: I'd break this sentence into at least two, e.g., "...can be summarized briefly as follows:..." [new sentence]
line 368: "documented previously" in Germain & Laurin (2009)? Please add ref.
line 386-90: "established consensus" should come with a list of references behind the branching pattern and divergence estimates (ahh I see this is from line 396). Please also add these to the fig. 2 caption. Also "molecular divergence dates" are themselves contigent upon paleontological calibrations, and hopefully you've picked estimates that do not recycle other clock dates as calibrations themselves (see Graur & Martin 2004, Reisz & Muller 2004, both in Trends Genetics).
line 415: as you've assembled a (very impressive) ossification dataset across osteichthyans, data from birds and mammals are relevant. However stating here with "for the birds" is a bit sudden, and you might add a sentence here to remind readers the importance of amniote data for your study of lissamphibian origins. Also I'm not sure about the topologies in Pons et al., Wang et al & Gonzales et al., but the Prum et al. 2015 (very large) dataset shows topological conflict with other, large genomic studies, in particular Jarvis et al. 2014 reflecting what remains a stubborn polytomy at the base of Neoaves (nicely reanalyzed & discussed in Reddy et al. 2017 Syst Bio). How might the competing phylogenies in (say) Prum et al. 2015 vs Jarvis et al. (2014) influence your interpretations of amniote ossification sequences?
line 419: A good summary & rationale for mammalian divergences (and why some estimates may be too old) is Phillips & Fruciano 2018 BMC Ev Biol, also Dos Reis et al. 2012 (Proc Roy Soc B). A good compendium of vertebrate divergence dates in Benton et al. 2015 Paleont Electr.
line 477 (and elsewhere): paragraphs like this that have frequent references to acronyms (DH, LH, PH2, etc.) are hard to follow. It's fine to shorten the text w/ such acronyms but perhaps you could add parentheticals to remind your readers that "PH2" etc. are shown in your fig. 1a, b, c, etc.
line 516: This sentence would be easier to follow if you broke it up, e.g., rephrase text from "but it is weaker..." as a new sentence. Ditto for the long sentence in lines 518-523.
The Fig. 1 caption is very long. Perhaps move a few qualitative phrases (e.g., "very cautiously Froebisch et al. 2017...") to the main text. The detailed attributions of which authors are associated with which trees are important, but again could probably be moved to the main text while still making reference to those details with a single phrase in the caption, e.g., "See Methods for details on support for these competing topologies".
Fig. 2 caption is too short & makes no mention of the data behind this topology or divergence estimates. Please provide citations to make the caption self-contained and enable your readers to know the data & publications behind this tree. Please state what the horizontal colored lines represent (I guess marine stages?). Also they're garish and make the branches harder to read compared to (for example) grayscale, dotted lines, or similar.
line 1041: Just write "are" rather than "appear to be" (also line 1049). Again this caption has interpretation & detail (e.g., "...there is clearly a phylogenetic signal...") that is more appropriate for Results or Discussion than a figure caption.
line 1044: As noted previously RE: suppdata, write out what "SM 1" means (also line 1050) and add a statement (somewhere) indicating where this can be downloaded.
Appendix 1: without lines to indicate columns & rows, and without a repeating header at the top of each page, this appendix is hard to read. I would recommend moving this to online supplementary data in the form of a spreadsheet. Alternatively, reduce the font size and make this a table or figure with repeating a header and lines to delineate cells.
Recommender comments on Laurin et al. PCI-Paleontology by Robert Asher
22 Aug 2018 I've just got a third review in today and will make that available to the authors. (I don't think it shows up yet on the PCI-Paleo site). My comments below were written after having just the first two reviews; the third doesn't change my decision to "recommend revision", but does provide further constructive critiques that the authors should consider. My editorial comments are pasted below.
21 Aug 2018
Overall I like this manuscript and am keen to see it as a formally accepted paper in PCI-Paleontology. Both reviewers raise a number of issues which need to be addressed. R2 in paricular argues that taxon and character sampling is not quite sufficient to reject all hypotheses besides monophyletic origin among lepospondyls, or at least not as strongly as the authors do in this manuscript. I welcome a revision taking these critiques into account, and if possible increasing the scope of taxa and/or characters sampled as per reviewer critiques. Additional, minor comments of my own are pasted below. Please respond to all of these and the reviewer comments in your revision.
In the caption for fig. 1 write out the "LH" abbreviation (and wherever possible minimize acronyms in the text)
line 103: depending on your response to the reviewers, and given that you're looking at cranial sequences, it would be more informative here to note "...extensive database on cranial ossification sequences..."
line 111: state what the software was.
line 122: Characters missing for a given fossil, but present in other taxa, can have an impact on phylogenetic estimation for that fossil by one or both of the following interrelated effects: - changing placement of taxa to which the fossil may be related - changing number of steps (in a parsimony context) on a given tree of other characters that are known for that fossil. Given the above (detailed in Asher et al. 2005 JVP 25(4):911-923), are you sure that characters "could not be scored for the temnospondyls Apateon and Sclerocephalus, so they could not have helped resolve the main question examined in this study"?
line 144: I don't think many readers will remember these acronyms, but I recognize the benefit of not having to write each one out at every occurrence. Perhaps add more frequent references to your fig. 1, for example here, and remind readers that acronyms are defined & figured in your fig. 1.
line 168: It would be straightforward to apply an optimality criterion to these sequence data and actually test if they are indeed "unlikely to provide a well-resolved tree". You wouldn't need to figure anything or write at length, but note simply that---assuming you're correct--- method X (e.g., parsimov or others you prefer) "...yields an unresolved tree so instead we tested likelihoods of the competing hypotheses in Fig. 1 ..."
The parenthetical on lines 186-187 sounds a bit too informal & personal and I'd recommend deleting it.
line 203: replace "consensual" with something like "consensus" with relevant citations of the papers/phylogenies behind this consensus.
line 209: I think the term "databases" is more familiar written as one word.
line 234: Fabre et al. 2012 (BMC Evol Biol) present a well-sampled rodent phylogeny that includes both P.melanophrys and M.auratus; ensure that Wilson & Reeder 2005--- a more taxonomic than phylogenetic reference--- are consistent with their estimate.
line 243: this may seem like a trivial point but it's quite important: unless you're interested in gene trees, phylogenies aren't "molecular" or "morphological" but rather the data used to reconstruct them can entail one or both. So write "molecular phylogenetic analysis" or "... most recent phylogenies based on genomic/molecular data" rather than "molecular phylogenies" (as you've done elsewhere in the manuscript, e.g., lines 15, 50, 82 ...)
line 245: can you better justify your disagreement with Irisarri et al. 2017 (or cite someone to this effect)?
line 261: you might add an "and" before "in case", and/or better explain the connection between the "continuous evolutionary model" and why equal branch lengths are important to minimize bias against a particular hypothesis.
line 266: remind readers why inclusion/exclusion of Sclerocephalus & the squamosal is relevant here.
line 291: "previous attempts" sounds pejorative, and agree or not, Anderson's conclusions are not simply "attempts". So delete "attempts" and just write "previous phylogenetic conclusions from ossification sequences..."
line 299: by "untenable" you mean broadly regarded as false, as in amniote or mammalian non-monophyly, right? Please clarify.
line 356: I'd start a new sentence after "LH" and delete "especially" & start the new sentence with "Similarities ..."