Palatal morphology predicts the paleobiology of early salamanders

  1. Jia Jia  Is a corresponding author
  2. Guangzhao Li
  3. Ke-Qin Gao  Is a corresponding author
  1. School of Earth and Space Sciences, Peking University, China
  2. State Key Laboratory of Palaeobiology and Stratigraphy (Nanjing Institute of Geology and Palaeontology, CAS), China
  3. Department of Comparative Biology and Experimental Medicine, University of Calgary, Canada
  4. Department of Microbiology, Immunology, and Tropical Medicine, The George Washington University, United States

Abstract

Ecological preferences and life history strategies have enormous impacts on the evolution and phenotypic diversity of salamanders, but the yet established reliable ecological indicators from bony skeletons hinder investigations into the paleobiology of early salamanders. Here, we statistically demonstrate by using time-calibrated cladograms and geometric morphometric analysis on 71 specimens in 36 species, that both the shape of the palate and many non-shape covariates particularly associated with vomerine teeth are ecologically informative in early stem- and basal crown-group salamanders. Disparity patterns within the morphospace of the palate in ecological preferences, life history strategies, and taxonomic affiliations were analyzed in detail, and evolutionary rates and ancestral states of the palate were reconstructed. Our results show that the palate is heavily impacted by convergence constrained by feeding mechanisms and also exhibits clear stepwise evolutionary patterns with alternative phenotypic configurations to cope with similar functional demand. Salamanders are diversified ecologically before the Middle Jurassic and achieved all their present ecological preferences in the Early Cretaceous. Our results reveal that the last common ancestor of all salamanders share with other modern amphibians a unified biphasic ecological preference, and metamorphosis is significant in the expansion of ecomorphospace of the palate in early salamanders.

Editor's evaluation

This paper is a valuable contribution to evolutionary ecomorphology in extant and extinct tetrapods, and of interest to vertebrate paleontologists and other evolutionary biologists interested in the early evolution of amphibians. Using geometric morphometric analysis, the authors demonstrate that both the shape of the palate and several non-shape variables (particularly associated with vomerine teeth) are ecologically informative in early stem- and basal crown-group salamanders. The study also reveals that metamorphosis is significant in the expansion of ecomorphospace of the palate in early salamanders.

https://doi.org/10.7554/eLife.76864.sa0

Introduction

Salamanders, anurans, and caecilians are highly distinctive from one another in their morphology in both living species and their respective oldest known relatives from the Triassic (Ivachnenko, 1978; Ascarrunz et al., 2016; Pardo et al., 2017a; Schoch et al., 2020; Kligman et al., 2021). As a result, the evolutionary origin(s) of modern amphibians have remained controversial since the late 19th century (Haeckel, 1866), with a number of extinct tetrapod groups in different ecological types at adult stages having been hypothesized as their purported ancestors, including: amphibamid (terrestrial) and branchiosaurid (terrestrial and aquatic) dissorophoid temnospondyls (Laurin et al., 2004; Fröbisch and Schoch, 2009; Maddin and Anderson, 2012; Pardo et al., 2017b), stereospondylian (semiaquatic/aquatic) temnospondyls (Schoch and Milner, 2014; Pardo et al., 2017a), and several groups of lepospondyls (aquatic, semiaquatic or terrestrial; Marjanović and Laurin, 2013; Marjanović and Laurin, 2019; Jansen and Marjanović, 2021; Laurin et al., 2022). The specialized morphologies in modern amphibians are greatly impacted by ecology and their complex life history strategies (e.g. Wake, 2009), for example, even the earliest anuran Triadobatrachus and the possible caecilian Chinlestegophis from the Triassic display several morphological specializations as their living relatives for aboveground and subterranean terrestrial living settings, respectively. Salamanders (or Caudata, the total group), on the other hand, have a more conservative body plan and more diversified ecological preferences when compared to anurans and caecilians (Deban and Wake, 2000; Bonett and Blair, 2017; Fabre et al., 2020), and have been frequently used as comparative analogues for inferring the paleoecology of extinct tetrapods (Schoch and Fröbisch, 2006; Fröbisch and Schoch, 2009). However, the evolutionary paleoecology in modern amphibians and particularly in early salamanders has received insufficient attention.

Cryptobranchoidea is the sister group of all other crown group salamanders (Urodela) and contains two subclades: Pancryptobrancha (total group cryptobranchids; Vasilyan et al., 2013) and Panhynobia (total group hynobiids; Jia et al., 2021a). The two subclades are united by a set of synapomorphies (Dunn, 1922; Estes, 1981; Jia et al., 2021a), but are different from each other in life history strategies and ecological preferences at their respective adult stage: most pancryptobranchans are neotenic or partially metamorphosed and live in water permanently by retaining larval features (e.g. gill slits), albeit the pancryptobranchan Aviturus from the Paleocene was interpreted as semiaquatic with an unknown life history strategy (Vasilyan and Böhme, 2012; but see Skutschas et al., 2018). In contrast, panhynobians are predominantly metamorphosed, except that the stem hynobiid Regalerpeton from Early Cretaceous (Rong, 2018) and some populations of the living hynobiid Batrachuperus londongensis are neotenic (Jiang et al., 2018). Postmetamorphosed hynobiids have lost larval features and are characterized by an anterolaterally directed palatal ramus of the pterygoid, and are able to live in water (e.g. Paradactylodon), on land (e.g. Hynobius) or are semiaquatic (Ranodon) outside of the breeding season (Kuzmin and Thiesmeier, 2001; Fei et al., 2006; Materials and methods).

Cryptobranchoidea are critical in understanding the paleoecology of early salamanders because the earliest known cryptobranchoids from the Middle Jurassic (Bathonian) have higher disparities in both life history strategies and ecological preferences than stem urodeles, and represent the oldest known crown urodeles, including ‘Kirtlington salamander B’ from the UK, Kiyatriton krasnolutskii from Russia, and Chunerpeton, Neimengtriton, and Jeholotriton from China (Evans and Milner, 1994; Gao et al., 2013; Skutschas, 2015; Jia et al., 2021a). Both Chunerpeton and Jeholotriton are neotenic as confirmed by the presence of external gills and a tall caudal dorsal fin in adult specimens (Gao and Shubin, 2003; Wang and Rose, 2005), whereas Neimengtriton is the oldest metamorphosed and semiaquatic cryptobranchoid (Jia et al., 2021a; see below). The ‘Kirtlington salamander B’ and K. krasnolutskii are both represented by fragmentary materials and their paleoecology unfortunately remains unknown. In contrast, other contemporaries (e.g. Kokartus, Marmorerpeton) from the Middle Jurassic (Bathonian) of UK, Russia, and Kyrgyzstan are all neotenic and aquatic at their adult stage, and have been classified as stem urodeles by the absence of spinal nerve foramina in the atlas that characterizes Urodela (Ivachnenko, 1978; Evans et al., 1988; Skutschas and Krasnolutskii, 2011; Skutschas and Martin, 2011; Skutschas, 2013; Skutschas et al., 2020). The only known pre-Jurassic stem urodele, Triassurus from the Middle/Upper Triassic of Kyrgyzstan, is merely represented by two larval specimens with no clue to its paleoecology at adult stage (Schoch et al., 2020).

To date, seven other basal cryptobranchoids have been reported from the Upper Jurassic to Lower Cretaceous of northern China: Laccotriton, Liaoxitriton, Linglongtriton, Nuominerpeton, Pangerpeton, Regalerpeton, and Sinerpeton, most of which are represented by articulated specimens and have been recently recovered as stem hynobiids or hynobiid-like taxa (see Gao et al., 2013; Jia and Gao, 2016; Jia and Gao, 2019; Jia et al., 2021a). Besides the neotenic Regalerpeton as aforementioned, habitat preferences of these metamorphosed taxa and paleoecological disparity patterns of Cryptobranchoidea remain largely unexplored mainly due to yet established osteological indicators for ecology (see Discussion). The configuration of vomerine teeth has long been identified as useful for the classification of living cryptobranchoids (Zhao and Hu, 1984) and was recently claimed to be ecologically informative (Jia et al., 2021b), but such statements have not received rigorous tests with inclusion of fossil taxa.

Our series of studies on living and fossil cryptobranchoids noticed that besides the vomerine teeth, the palate varies in shape and proportion, and could potentially serve as an indicator for paleoecological reconstruction (Jiang et al., 2018; Jia et al., 2019; Jia et al., 2021b; Figure 1 and Figure 1—figure supplements 123). To test these hypotheses and to address the constraints underlying the morphological disparity of the palate, here we conducted a 2D landmark-based geometric morphometric analysis on the palate of all living and most aforementioned fossil genera of cryptobranchoids, stem, and other basal crown urodeles based primarily on micro-CT scanned specimens. We statistically investigated disparity patterns within the morphospace of the palate with respect to ecological preferences, life history strategies, and taxonomic affiliations. Based on a time-calibrated cladogram we established for fossil and living cryptobranchoids (Jetz and Pyron, 2018; Jia et al., 2021a), we further quantified the evolutionary rate of the palate and reconstructed the ancestral states for ecological preferences, life history strategies, palate shape, and vomerine tooth configurations of the respective last common ancestor of Panhynobia, Pancryptobrancha, Cryptobranchoidea, Urodela, and Caudata. We demonstrate that the palate is a reliable proxy in ecological reconstructions for early salamanders, and the morphospace of the palate is predominantly shaped by ecological constraints and also displays a stepwise evolutionary pattern.

Figure 1 with 26 supplements see all
The palate and phylogenic relationships of early salamanders.

(A) The vomer (gold) and parasphenoid (purple) of the palate in ventral view of the skull in living hynobiid Pseudohynobius flavomaculatus. (B) Dorsal view of the palate showing the articulation patterns with the paired orbitosphenoid (whitish). (C) Enlarged view of the palate in ventral view with red circles corresponding to the 24 landmarks used for the geometric morphometric analysis. (D) Palatal configurations of early salamanders in ventral view, with color-coded life history strategies (square block) and ecological preferences (line) plotted on the time-calibrated tree modified from Jetz and Pyron, 2018 and Jia et al., 2021a.

Results

In ventral view of the palate, the anteromedial fenestra is present between the vomer and the upper jaw in most early salamanders and is only absent in living cryptobranchids (Figure 1—figure supplements 123). The paired vomers medially articulate with each other in most taxa and posteriorly overlap to different extents, the anterior part of the cultriform process of the parasphenoid and/or the orbitosphenoid. The teeth are closely packed as a continuous tooth row positioned along the anterolateral periphery of the vomer in cryptobranchids but have diversified configurations in other taxa. The parasphenoid is a sword-like, azygous bony plate with its anterior part articulating dorsally with the orbitosphenoid and its posterior part flooring the otic capsule.

Morphospace and shape disparity patterns of the palate in early salamanders

The palate is symmetric about the mid-sagittal plane of the skull with symmetric shape components accounting for 96.15% of the total shape variation in 70 specimens, and the left-right asymmetry accounting for the remaining 3.85%. The shape and the size of the palate with the latter represented by the centroid size (CS), are significantly correlated as revealed by the standard multivariate regression between log (CS) (independent variable) and symmetric shape components (dependent variable) across 70 specimens (R2 = 9.3331%; p<0.001; F = 6.9977; Z = 3.918) and 34 species (R2 = 13.276%; p<0.001; F = 4.8985; Z = 3.1691). However, when phylogenetic relationships of the 34 species were factored in, the association between size and shape of the palate is no longer significant as shown in the evolutionary allometry analysis (p=0.1583). Such inconsistency between standard and evolutionary allometry analyses is related to the fact that the CS of the palate has a strong phylogenetic signal (Blomberg’s K = 0.997, p=0.001, Z = 4.1921) and, hence, the CS accounts for an even smaller amount of shape variations of the palate (R2 = 4.494%; F = 1.5059; Z = 1.0141) when evolutionary history among species was counterbalanced in the evolutionary allometry analysis. To eliminate impacts from both asymmetry and allometry on the spatial patterns of the palate, residuals from the multivariate regression of symmetric shape components on log (CS) were retained for downstream statistical analyses.

To visualize the spatial patterns of the palate in the morphospace, on the basis of the size-corrected (allometry-free) 24-landmark dataset, we conducted a standard principal component analysis (PCA) across 70 specimens; and we also conducted three other types of PCA across 34 species with the time-calibrated cladogram and ancestral internal nodes projected into the morphospace (Figure 2—figure supplements 13), including: a phylomorphospace analysis (PA), phylogenetic principal component analysis (Phylo-PCA) and a phylogenetically-aligned components analysis (PaCA). The first three PC axes in each of the four PCAs collectively measure up to about 70% of total shape variances (Supplementary file 1A). Within the phylomorphospace defined by principal components (PCs) 1–2 (Figure 2B and Figure 2—figure supplement 1b), aquatic and terrestrial living species of Cryptobranchoidea are generally located along the positive and negative interval of the PC 2 axis (21.35%), respectively. Most taxa are ecologically exclusive at the genus level, except that Liua and Paradactylodon are the only two living hynobiid genera with species occupying both the aquatic (Liua shihi and Paradactylodon mustersi) and terrestrial (Liua tsinpaensis and Paradactylodon persicus) zones. The living semiaquatic hynobiid Ranodon sibiricus occupies at a location intermediate between the aquatic and terrestrial zones. Shape changes of the palate relative to the mean shape of all terminal and internal taxa along the positive values of PC 2 (aquatic zone) involve an anteromedial extension of the vomer and therefore a reduction of the anteromedial fenestra, a shrinkage of the anterolateral and posterolateral borders of the vomer and the width of the cultriform process of the parasphenoid, and an anterior extension of the parasphenoid. In contrast, the negative values of PC 2 (terrestrial zone) characterize a shrinkage of the anteromedial border of the vomer and therefore a posterolaterally expanding anteromedial fenestra, a laterally widening retrochoanal process of the vomer and the cultriform process of the parasphenoid, and an anteroposteriorly shortening parasphenoid. On the other hand, basalmost crown urodeles (e.g. Beiyanerpeton, Chunerpeton, and Pangerpeton) and karaurids are largely separated from living taxa along the PC 1 axis (37.49%), with stem hynobiids widely scattered in the phylomorphospace and lying within either the aquatic (Liaoxitriton) or terrestrial (Linglongtriton and Nuominerpeton) zone. The only known aquatic stem hynobiid Regalerpeton is situated at a region of the aquatic zone occupied by other aquatic fossil taxa (e.g. Beiyanerpeton and Chunerpeton), whereas the semiaquatic stem hynobiid Neimengtriton occupies a location closer to terrestrial than either aquatic zone or the semiaquatic zone of extant hynobiids. From the largest to the smallest value of PC 1, both the vomer and the parasphenoid have an anteroposterior extension and the cultriform process of the parasphenoid changes from an anteriorly widened plate with an indented anterior edge into a bilaterally narrowed plate with a pointed anterior edge.

Figure 2 with 5 supplements see all
Spatial patterns of the palatal shape in the morphospace defined by the first two components generated from four principal component analyses (PCA).

(A) Standard PCA across 70 specimens, (B) phylomorphospace analysis, (C) phylogenetically aligned components analysis, and (D) phylogenetic PCA across 34 species with ancestral states for internal nodes (open circles) and phylogenetic relationships (black lines) plotted in the morphospace. The color and shape of each point represent the ecological type and taxonomic affiliation, respectively. Extreme values of the palatal shape along both principal components (PCs) 1 and 2 are represented by wireframes color-coded to ecological types against the mean shape (gray) of both terminal and internal taxa.

The evolutionary history among species has a moderate but significant contribution in the formation of the spatial patterns in the phylomorphospace (phylogenetic signal: observed Kmult = 0.4154, p=0.001, Z = 4.9856). When the multivariate shape data of the palate were maximally aligned with phylogenetic signal (Figure 2C and Figure 2—figure supplement 1c), fossil taxa can be roughly divided from living taxa along PaCA-C 1. Both crown and stem taxa of Panhynobia are more compactly clustered than that seen in the phylomorphospace created by PA, and are distinct from the Pancryptobrancha and the region occupied by karaurids, basal cryptobranchoids and salamandroids; but taxa in different types of ecological preference are mixed together. By contrast, when the phylogenetic signals of the palate were eliminated by the Phylo-PCA (Figure 2D and Figure 2—figure supplement 1d), the overall spatial patterns among species are essentially preserved, albeit slightly rotated clockwise, as observed in the phylomorphospace. In this phylogeny-free morphospace, the last common ancestors of Pancryptobrancha and of Hynobiidae lie within the aquatic zone and are tightly associated with living cryptobranchids and the stem urodele Kokartus, respectively. In contrast, the last common ancestors of Panhynobia, Cryptobranchoidea, Urodela, and Caudata lie within the terrestrial zone and are adjacent to three highly derived living hynobiids, respectively, Pseudohynobius shuichengensis, Pseudohynobius jinfo, and Liua tsinpaensis.

Our pairwise comparison (Supplementary file 1B–E) and phylogenetic Procrustes ANOVA reinforce that the shape of the palate is significantly different among groups of species that are classified by ecological preference (R2 = 28.079%; p=0.001; F = 4.3740; Z = 3.4838) and life history strategy (R2 = 6.797%; p=0.006; F = 3.1766; Z = 2.4673), but not by taxonomic affiliations, neither at the genus (R2 = 68.711%; p=0.248; F = 1.2549; Z = 0.78492) nor family (R2 = 12.858%; p=0.581; F = 0.8263; Z = –0.21234) level. With regard to the life history strategy, living cryptobranchids are separated from all Mesozoic neotenic taxa including Beiyanerpeton, Chunerpeton, karaurids, and Regalerpeton by the vast majority of the metamorphosed taxa, which occupy most of the morphospace of the palate (Figure 2—figure supplement 4). The single living neotenic hynobiid B. londongensis is situated between the two neotenic groups as aforementioned and lies alongside its metamorphosed conspecifics and those all collectively overlap with other aquatic hynobiids. When the seven-landmark-dataset for the right vomer was analyzed following the same procedure, the purported semiaquatic basal pancryptobranchan Aviturus is nested within the terrestrial zone along with living metamorphosed taxa (Figure 2—figure supplement 5).

Evolutionary rates of the palate

The palate exhibits considerable differences in morphological disparity and evolutionary rates across regions represented by the 24-landmark points as calculated under a Brownian motion evolutionary model (Figure 3; Supplementary file 1B–H). The vomer evolves about two times faster than the parasphenoid, and such a pattern is also supported by the fact that the vomer has a higher phylogenetic signal (Kmult = 0.6407) than that of the palate (Kmult = 0.4154). The posterior border of the vomer and the medial-most part of the choanal notch are the fastest and the slowest evolving parts in the palate, respectively. In the parasphenoid, however, the highest evolutionary rates are concentrated on the anterior end, posterior end and the lateral alae, and the lowest evolutionary rates are located at the posterior contact between the cultriform process and the orbitosphenoid and the junction area where the cultriform process merges with the lateral alae.

Figure 3 with 2 supplements see all
Evolutionary patterns of both the shape and non-shape covariates of the palate and their association with ecological disparity in early salamanders.

Ancestral shape (wireframes color-coded to ecological types superimposed with mean shape [gray]) and vomerine tooth row (zigzag black lines) configurations are reconstructed for respective last common ancestors of Hynobiidae, Panhynobia, Pancryptobrancha, Cryptobranchoidea, Urodela, and Caudata. A complete list of evolutionary rates for each of the 24 landmarks, the vomer, parasphenoid, the palate and the continuous covariates of the vomer across 34 species is available in Supplementary file 1GH and J. The two pie charts at each internal node of the time-calibrated cladogram are likelihoods of the position (left) and arrangement (right) of the vomerine tooth row reconstructed in this study. Continuous covariates of the vomer were subjected to Mann-Whitney U test for their association with the three ecological groups with corresponding p values labeled above the boxplots.

Species in different ecological (p=0.002, Z = 2.6284) and taxonomic (p=0.035, Z = 1.7594) groups vary greatly in evolutionary rate of the palate (Supplementary file 1H). Namely, aquatic and terrestrial taxa are comparable with each other in the evolutionary rate of the palate, the vomer and the parasphenoid, and both of them evolve at a rate that is less than half of that in semiaquatic taxa championed by Neimengtriton. Stem hynobiids have the highest evolutionary rates, followed successively by basal cryptobranchoids, pancryptobranchans, crown hynobiids, and non-cryptobranchoid basal salamanders. Neotenic and metamorphosed taxa are not significantly different from each other in evolutionary rates (p=0.843, Z = –1.1341).

Non-shape covariates from the vomer

Similar to shape variables, allometry has a significant impact on the first 4 of the 5 continuous non-shape covariates of the palate (length ratios between parasphenoid and palate, and that between vomer and palate; width ratios between vomerine tooth row [VTR] and vomer, and between outer and inner branches of the VTR; and vomerine tooth number) when each of them is statistically regressed on the log (CS). We thus use residuals from the regression for boxplots to visualize the distribution patterns of the covariates in three ecological groups across 70 specimens, and use the Mann-Whitney U-test to analyze if the covariates are reliable ecological indicators (Figure 3). In line with shape changes revealed from PCA, aquatic and terrestrial species are significantly different in all of the five covariates, with aquatic taxa having proportionally a longer parasphenoid (median: ~0.84), a shorter vomer (median: ~0.28), a higher ratio in outer/inner branch of the VTR (median: ~1.6), a narrower VTR (except neotenic taxa; median: ~0.37) and fewer vomerine teeth (median: 7) than that of terrestrial taxa (i.e. ~0.78, ~0.33, ~0.58, ~0.64, and 16). Semiaquatic species cannot be confidently differentiated from aquatic or terrestrial taxa in any of the five covariates, but interestingly their parasphenoid length and VTR width resemble those in terrestrial taxa and their vomer length, outer/inner branch VTR width ratio and teeth numbers are more similar to aquatic taxa. The contingency table and Cochran-Mantel-Haenszel statistical test (Figure 3—figure supplement 1 and Supplementary file 1I) show that the arrangement and position of VTR are both significantly correlated to ecological preference and life history strategy. The VTR in anterior and middle position of the vomer or being arranged obliquely or parallel to the marginal tooth row are only present in aquatic taxa, with the VTR located anteriorly exclusively present in neotenic species. The VTR in the mid-posterior position of the vomer or when it is transversely arranged is only present in metamorphosed taxa with all kinds of ecological preferences. Species with VTR in the posterior position or transversely arranged are more likely to be terrestrial than aquatic or semiaquatic.

Ancestral states of the palate

The ancestral states in both the shape and non-shape covariates of the palate were reconstructed for the last common ancestors of Hynobiidae, Panhynobia, Pancryptobrancha, Cryptobranchoidea, Urodela, and Caudata, respectively, using maximum likelihood (Supplementary file 1J). When compared to the mean shape of the palate, the configurations of the palate in the respective last common ancestors of Caudata, Urodela, and Cryptobranchoidea are characterized by a shortened parasphenoid with an anteriorly widened cultriform process and an anteriorly shortened but posteriorly widened vomer. This configuration coincides with our reconstructions of the respective last common ancestors of Caudata, Urodela, and Cryptobranchoidea is parallel to the marginal tooth row and is located in the posterior part of the vomer with the outer longer than the inner branch. The VTR retained a similar configuration, except for being transversely arranged in the last common ancestors of Panhynobia and Pancryptobrancha and then shifting to the middle part of the vomer in the last common ancestor of Hynobiidae.

Discussion

Among modern amphibians, anurans, and caecilians undergo metamorphosis or direct development, and the adults of most species are terrestrial. In contrast, salamanders have their ecological preference decoupled from life history strategy, especially in metamorphosed taxa such as living and extinct hynobiids where postmetamorphosed adults live in water, on land or are semiaquatic (Fei et al., 2006; Jia and Gao, 2016; Jia et al., 2021a). The discrepancy in ecological preference between salamanders on one hand and anurans and caecilians on the other hand is unhelpful in understanding the evolutionary paleoecology in the early lissamphibians given that salamanders and anurans are sister-groups. Previous studies on fossil salamanders generally focus on the taxonomy, whereas the paleoecology and the evolutionary history of the ecological decoupling particularly in metamorphosed taxa had received insufficient attention, which is to some extent explained by the insufficient taphonomic analyses on fossil sites with salamander discoveries, such as the Daohugou fossil locality (Wang et al., 2019). The main obstacles are (1) sufficient and reliable ecological indicators in the bony skeleton have not yet been established for extant cryptobranchoids and early salamanders (Xiong et al., 2016; Jia et al., 2021b), and (2) soft anatomical structures (e.g. labial fold and caudal fin) and stomach contents that are ecologically informative, as commonly seen in lacustrine deposits of certain neotenic species (Dong et al., 2011), are rarely preserved in metamorphosed individuals. Here, we quantitively investigate the paleoecological turnover in the earliest known salamanders based on the shape and non-shape variables of the palate. Our results shed light on the interactions between morphology and paleoecology, and the underlying mechanisms governing ecomorphological diversity among early salamanders along the rise of modern amphibians.

As shown in the phylomorphospace, the shape of the palate in early salamanders is heavily impacted by convergence resulting from ecological disparity, as extant cryptobranchoids and fossil taxa in different ecological preferences occupy distinctive zones in the morphospace. Such spatial patterns remain essentially unaltered after phylogenetic signals are eliminated by the Phylo-PCA, along the first axis of which aquatic and terrestrial taxa regardless of their phylogenetic closeness are separated from each other, confirming our hypothesis that the shape of the palate is a reliable ecological indicator in early salamanders as reported in living salamanders (Fabre et al., 2020). As shown above, non-shape covariates of the palate particularly with regard to the VTR are also ecologically informative. The palate in aquatic species is characterized by a reduction of the anteromedial fenestra, a shrinkage of the vomer laterally and anteroposteriorly with fewer vomerine teeth, a high ratio of the outer to the inner branch of the VTR, a narrow VTR, and an elongated parasphenoid. On the other hand, the palate in terrestrial species is characterized by a posterolateral expansion of the anteromedial fenestra, an anteroposteriorly elongated vomer and a widened retrochoanal process with more vomerine teeth, a low ratio of the outer to inner branch of the VTR, a widened VTR, and an anteroposteriorly shortened parasphenoid. The palate in extant semiaquatic taxa is transitional in both the morphospace and many non-shape covariates between aquatic and terrestrial taxa as mentioned above, whereas the palate of the only known fossil semiaquatic salamander, Neimengtriton (Middle Jurassic) is configured more like that of terrestrial taxa as argued in the original study (Jia et al., 2021a), indicating that Neimengtriton is probably more adapted to terrestrial environments despite being semiaquatic. The respective last common ancestors for Caudata, Urodela, Cryptobranchoidea, and Panhynobia, the basal cryptobranchoid Pangerpeton (Late Jurassic) and the stem hynobiids Linglongtriton (Late Jurassic) and Nuominerpeton (Early Cretaceous) are terrestrial, whereas other stem hynobiids Liaoxitriton and Regalerpeton (Early Cretaceous) and the respective last common ancestors for Hynobiidae and Pancryptobrancha are aquatic, demonstrating that ecological shifts occurred frequently in the evolution of early salamanders. The Paleocene pancryptobranchan Aviturus is likely metamorphosed and terrestrial based on the shape and non-shape variables of the vomer, but this hypothesis awaits to be tested by the discovery of a more completely preserved palate.

Morphological adaptations in the palate and perhaps in other cranial features (e.g. hyobranchial apparatus; see Jia et al., 2021b) to aquatic and terrestrial living settings are likely constrained by feeding mechanisms (Regal, 1966; Reilly and Lauder, 1990). In extant cryptobranchoids, terrestrial species grasp prey items with their jaws and/or by their tongue protruding from the mouth; the captured prey is then transported from the snout into the esophagus with the assistance of several cyclical movements of tongue and hyobranchial apparatus that press and reposition the prey against the palate (Deban and Wake, 2000; Wake and Deban, 2000). Such feeding mechanisms in terrestrial cryptobranchoids demand a sticky tongue moisturized by the intermaxillary or internasal gland that is housed above an expanded anteromedial fenestra, a wide snout contributed by lateral expansions of the vomer, many vomerine teeth to efficiently hold the prey in place, and a shortened parasphenoid to reduce the distance of intraoral transportation. The posteriorly elongated inner branch of the VTR as represented by derived terrestrial hynobiids Hynobius and Salamandrella would serve as toothed surfaces that can further facilitate tongue manipulations and transportations of small prey items posteriorly as in more sophisticated terrestrial feeders (e.g. salamandrids and plethodontids; Regal, 1966). By contrast, aquatic salamander species capture their prey primarily by suction feeding in which the prey is carried into the biting range of the jaws by water currents created by retraction and expansion of the buccal walls, and giant salamanders can even perform asymmetrical strikes in water via unilateral jaw and hyobranchial movements (Gillis and Lauder, 1994). Intraoral transport of the prey is more efficient in aquatic than in terrestrial species; and it typically involves repeating the same procedure as in the initial suction strike and is occasionally assisted by tongue manipulations against the palate (Regal, 1966). The limited usage of the tongue in aquatic prey capture diminishes the need for moisturization from the intermaxillary or internasal gland which, in turn, may lead to reduction and closure of the anteromedial fenestra as seen in aquatic species. An anteroposterior expansion of the parasphenoid increases buccal volume, and the shrinkage of the vomer along both the mediolateral and anteroposterior dimensions reduces snout size; both features act in concert to boost the success of feeding attempts by creating a high negative buccal pressure. A narrow VTR with few teeth, most of which are located on the outer branch, likely serves to reduce hindering the influx of water and prey.

Whichever mode of prey capture and intraoral transportation cryptobranchoids perform, the palate undergoes a similar mechanical stress distribution pattern as found in species with different types of ecological preference and life history strategy (Fortuny et al., 2015: Andrias davidianus; Zhou et al., 2017: Salamandrella keyserlingii). Such findings reinforce that alternative morphological configurations of certain features in the palate cope well with similar functional demands, but do not necessarily indicate that other feeding-related features of the palate and other regions of the cranium would also exhibit such extensive convergence. For example, the ceratohyal has been argued to be ecologically informative in extant cryptobranchoids, as the ceratohyal is consistently ossified at its posterior end in aquatic species but remains cartilaginous in terrestrial taxa (Xiong et al., 2016; Jia et al., 2021b); however, the ceratohyal remains cartilaginous in all known early fossil stem and crown urodeles, and ossifications of the ceratohyal must represent a derived feature independently evolved in crown cryptobranchoids (Jia et al., 2021b). Interestingly, the palate of extant cryptobranchoids as shown in biomechanical analyses (Fortuny et al., 2015; Zhou et al., 2017) has the highest stress level concentrated in the most posterior part of the vomer and its sutural area with the parasphenoid and orbitosphenoid, which surprisingly correspond to the place with the highest evolutionary rate in the palate as revealed by our study. Here, we recognize a clear stepwise evolutionary pattern at the vomer-parasphenoid-orbitosphenoid sutural area in early salamanders (Figure 4). In stem (Karaurus and Kokartus) and many basal crown (Beiyanerpeton, Chunerpeton, Jeholotriton, Neimengtriton, Pangerpeton, and Qinglongtriton) urodeles from the Jurassic and the reconstructed respective last common ancestors for Panhynobia, Cryptobranchoidea, Urodela, and Caudata, the vomer has a limited contact with the parasphenoid, the cultriform process of the parasphenoid is anteriorly expanded bilaterally, and the orbitosphenoid has no anteroventral processes (see below) or in the case of Qinglongtriton remains completely cartilaginous. In three stem hynobiids (Liaoxitriton, Nuominerpeton, and Regalerpeton) and the reconstructed ancestors for Hynobiidae and Pancryptobrancha from the Cretaceous and another stem hynobiid Linglongtriton from the Late Jurassic, the vomer has an increased contact with the parasphenoid, the cultriform process of the parasphenoid is anteriorly constricted, and the orbitosphenoid lacks the anteroventral process. In living cryptobranchoids, the vomer is in more extensive contact with the parasphenoid and orbitosphenoid (Trueb, 1993: Cryptobranchus; Kuzmin and Thiesmeier, 2001: Ranodon; Jiang et al., 2018: Batrachuperus; Jia et al., 2021b: Pseudohynobius), the cultriform process of the parasphenoid is anteriorly constricted bilaterally, and the orbitosphenoid has a thick anteroventral process ossified from neighboring cartilages in the nasal capsule that projects medially and firmly overlaps the cultriform process of the parasphenoid. If similar biomechanical stress patterns existed in early salamanders, the stress and strain applying to the vomer/parasphenoid sutural area would be equally offset by either a wide and thin cultriform process of the parasphenoid with limited support from the vomer and the absence of anteroventral process of the orbitosphenoid as seen in Jurassic taxa, or a narrow cultriform process thickened by more extensive overlapping with the vomer as seen in Cretaceous and living taxa, and by ossification of the anteroventral process of the orbitosphenoid as seen solely in living taxa.

Spatial-temporal patterns of phenotypic diversities of the palate and their associations with ecological preference and life history strategy in early salamanders and the stepwise evolutionary patterns at the sutural area of vomer, parasphenoid, and orbitosphenoid.

The morphospace of the palate is defined within a space formed by geological time scale (Z-axis) and principal components (PCs) 1 (X-axis) and 2 (Y-axis) derived from phylogenetic principal component analysis across 34 species. All silhouettes and images of salamander species are original.

It is worth emphasizing that the phenotypic diversity of the palate is variously affected by neoteny and metamorphosis. Neoteny is notable in producing convergent characters through truncation of normal developmental trajectories and its indirect role associated with constraints from the aquatic environment (Wiens et al., 2005). Most early stem and crown urodeles are neotenic (e.g. Chunerpeton and Kokartus) and their phenotypic diversity of the palate is truly confined to a restricted area in the spatial-temporal morphospace. However, extant neotenic cryptobranchids and the hynobiid B. londongensis bear an increased phenotypic diversity of the palate as compared to fossil neotenic taxa, and are separated from one another within the morphospace with the neotenic B. londongensis even overlapping with its metamorphosed conspecifics, indicating that constraints imposed by ecology have more influence than neoteny in the morphogenesis of the palate. The ecomorphospace of the palate is indeed greatly and rapidly expanded by metamorphosed taxa represented by basal cryptobranchoids and most stem hynobiids as these taxa have the highest evolutionary rate in the palate (Supplementary file 1H). The last common ancestors of Caudata, Urodela, and Cryptobranchoidea are reconstructed to be metamorphosed and terrestrial as evidenced by the shape and non-shape variables of the palate. Disparities of ecological preference among metamorphosed taxa must have taken place before the Middle Jurassic (Bathonian), because one of the oldest known crown urodeles, Neimengtriton, is metamorphosed and semiaquatic as evidenced by the presence of a low but pliable dorsal caudal fin at adult stage. Most of the current scope in the phenotypic diversity of the palate possessed by modern cryptobranchoids were achieved by Early Cretaceous. Our results rigorously show that the shape of the palate and many non-shape covariates particularly associated with vomerine teeth are reliable ecological indicators for paleoecology of early salamanders, and we demonstrate that metamorphosis with biphasic ecological preference (aquatic larvae + terrestrial adults) is not only the ancestral lifestyle in salamanders but also significant for the rise and diversification of modern amphibians.

Materials and methods

Experimental design, specimens, and palate

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Our study includes 60 wet specimens (preserved in formalin) that represent 25 living species (29% of Hynobiidae and 75% of Cryptobranchidae) in all 12 living genera of Cryptobranchoidea (Andrias, Batrachuperus, Cryptobranchus, Hynobius, Liua, Onychodactylus, Pachyhynobius, Paradactylodon, Protohynobius, Pseudohynobius, Ranodon, and Salamandrella), and one fossil skeleton for each of five stem hynobiids (Liaoxitriton zhongjiani, Linglongtriton daxishanensis, Neimengtriton daohugouensis, Nuominerpeton aquilonare, and Regalerpeton weichangense), one basal pancryptobranchan (Aviturus exsecratus), two basal cryptobranchoids (Chunerpeton tianyiense and Pangerpeton sinense), one basal salamandroid (Beiyanerpeton jianpingense) and two stem urodeles (Karaurus sharovi and Kokartus honorarius; Dataset in Dryad). Sirenids were not included into this study for the following four reasons: (1) their palate is extremely specialized and is patterned with an enlarged palatine that is absent in most other salamanders and patches of teeth densely arranged on palatine and vomer; (2) sirenids are the only herbivory salamanders with a complex three-dimensional chewing behavior (Schwarz et al., 2020), and their specially configured palate may receive biomechanical patterns different from other salamanders; (3) the palate is incompletely preserved in the earliest known sirenid taxon Habrosaurus from the latest Early Cretaceous (Gardner, 2003); and (4) the existence of a ~90 Ma fossil gap between Habrosaurus and the earliest known salamandroids (Beiyanerpeton and Qinglongtriton) greatly impede our understanding of their early evolution of the palate. To keep the gender of species names consistent with that of genus names as per ICZN codes, we replaced the feminine/masculine species ending (‘-is’) by corresponding neuter forms (‘-e’) for genus names (e.g. Nuominerpeton) ending in the neuter noun ‘herpeton’ or ‘ἑρπετόν’ in Greek as suggested in Rong et al., 2021. In this study, each species is represented by one to three specimens, except for the only facultatively neotenic cryptobranchoid B. londongensis where both neotenic and metamorphosed populations are each represented by three specimens. Most of the specimens are accessioned in the following seven institutional collections: Capital Normal University (CNU), Beijing, China; Chengdu Institute of Biology (CIB), Chengdu, Sichuan Province, China; Field Museum of Natural History (FMNH), Chicago, USA; Liupanshui Normal University (HNUL), Liupanshui, Guizhou Province, China; Peking University of Paleontological Collections (PKUP), Beijing, China; Zhejiang Museum of Natural History (ZMNH), Hangzhou, Zhejiang Province, China; and Zunyi Medical University (ZMU), Zunyi, Guizhou Province, China. For specimens that we were not able to examine firsthand, we used publicly available micro-CT scan data of 12 living cryptobranchoid specimens (Dataset in Dryad) from the MorphoSource platform (MorphoSource.org) and published images for five fossil taxa, including Aviturus (Vasilyan and Böhme, 2012: Figure 3), Chunerpeton (Gao and Shubin, 2003: Figure 1), Karaurus (Ivachnenko, 1978: Figure 1), Kokartus (Skutschas and Martin, 2011: Figure 9), and Regalerpeton (Rong, 2018: Figure 5).

Besides the CT scan data obtained from MorphoSource, four fossil and all living specimens were micro-CT scanned using the following three high-resolution CT scanners: a Nikon XT H 320 LC scanner in the Industrial Micro-CT Laboratory at China University of Geosciences (Beijing); a GE Phoenix v/tome/x 240kv/180kv scanner in the PaleoCT Lab at The University of Chicago; and a Quantum GX micro-CT Imaging System (PerkinElmer, Waltham, USA) at CIB (Dataset in Dryad). No filter was used because beam hardening artifacts were not encountered during CT scanning. The voxel size of the volume files generated from CT scans ranges between 14.52–87.32 μm (see detailed parameters in Dataset in Dryad). File processing including segmentation and rendering were accomplished by VG Studio Max (version 2.2; Volume Graphics, Heidelberg, Germany), and images in jpeg format showing the ventral view of the skull were exported for landmark acquisition after digital removal of the mandible and the hyobranchium from the virtual models.

The palate in adult specimens of cryptobranchoids consists of the partes palatinae of the premaxilla and maxilla, paired vomers and a single median parasphenoid. An independently ossified palatine is present in few fossil taxa and is absent in the remaining, and is therefore not included in this study as a compromise between taxa sampling and landmarks collection. The pars palatina of both the premaxilla and maxilla is a narrow bony ledge and invariably contributes to a small portion of the palate by posteriorly articulating with the vomer, and hence is not considered in this study (Figure 1A). Both the vomer and parasphenoid are dorsoventrally flattened bony plates and are homologous across species studied here, and thus are ideal for landmark collections for both living and fossil specimens, considering that the palate in all available fossil cryptobranchoids is dorsoventrally preserved. All anatomical terms used here follow Trueb, 1993 unless otherwise stated.

Cladograms

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For multivariate phylogenetic comparative analyses (evolutionary allometry analysis, phylogenetic signal analysis, Phylo-PCA, PA, phylogenetic alignment component analysis, and phylogenetic ANOVAs; see below), a maximum clade credibility tree (MCC; a target tree with maximum sum of posterior probabilities on its internal nodes) with 24 living taxa in our dataset was created by the software TreeAnnotator (version 1.10.4; Drummond et al., 2012) based on 10,000 random, time calibrated trees from a recent Bayesian posterior distribution analysis of living amphibians (Jetz and Pyron, 2018). To maximally match the terminal taxa in the MCC tree with our dataset, the living hynobiid Batrachuperus taibaiensis was eliminated from multivariate phylogenetic comparative analyses, considering that B. taibaiensis was recently synonymized to Batrachuperus tibetanus (Fei and Ye, 2016). The MCC tree was configured in Newick format and was read and visualized in the package ‘ape’ (version 5.5; Paradis and Schliep, 2019) for the software R (version 4.0.5; R Development Core Team, 2021). The remaining fossil taxa in our dataset with age ranges determined from published literature (Dataset in Dryad) and the Paleobiology Database (https://www.paleobiodb.org) were incorporated into this MCC tree based on a recent cladogram (Jia et al., 2021a: Figure 6) we constructed for living and fossil cryptobranchoids (Figure 1D). Adding fossil taxa increases the accuracy for multivariate phylogenetic comparative analyses and ancestral state reconstruction (see below; Soul and Wright, 2021), but will inevitably bring zero-length branches when the age of internal nodes was considered equal to that of its immediate oldest descendant, resulting in difficulties in multivariate phylogenetic comparative analyses. To circumvent this problem, any zero-length branch in the cladogram was treated to have a same branch length with its first none zero-length ancestral branch by using the ‘equal’ dating method in the ‘DatePhylo’ function from the R package ‘strap’ (version 1.4; Bell et al., 2015). We followed the suggestion from the ‘strap’ tutorial by setting the root length as 60 Ma by using the age difference between the oldest taxon in the cladogram (Kokartus; ~170 Ma) and the first older outgroup taxon known to date (Triassurus; ~230 Ma). Considering that the palate of Aviturus is only represented by the right vomer, the cladogram (Figure 1—figure supplement 24) including Aviturus was only used for the phylogenetic signal analysis and principal component analysis (PCA; see below) of the right vomer; whereas the cladogram without Aviturus was mapped onto the morphospace for a number of PCA and evolutionary rate analysis.

Data acquisition, geometric morphometrics, and symmetry and asymmetry

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The geometry of the paired vomers and parasphenoid is represented on the ventral view of the palate by 24 type I (intersections of biological structures) and type II (maximum of curvature) 2D landmark points (Figure 1B; Bookstein, 1991; Slice et al., 1996) digitized by using tpsUtil (64 bit; version 1.76; Rohlf, 2015) and tpsDig2 (version 2.31; Rohlf, 2015) based on images of 70 specimens (the 24-raw-landmark dataset thereafter; see above; Dataset in Dryad). Most specimens have completely-preserved palate except that the Paleocene A. exsecratus is known only by a right vomer (Vasilyan and Böhme, 2012: Figure 3). In order to investigate the paleobiology of A. exsecratus, we created a separate dataset with seven corresponding landmarks (#1–#7) for the right vomer across all 71 specimens (termed as the seven-raw-landmark dataset thereafter; Dataset in Dryad). To reduce measurement errors, specimens were arranged alphabetically by the name of the image files, then landmarks in each specimen were digitized by the same author (J.J.) following the same order (landmark 1–24; Supplementary file 1K). Procrustes coordinates (24-Procrustes-landmark dataset and seven-Procrustes-landmark dataset) for the geometry of the palate were obtained in the software MorphoJ (version 1.07 a; Windows platform; Klingenberg, 2011) after superimposing the raw landmark coordinates of all specimens through the Generalized Procrustes Analysis (GPA; Bookstein, 1991; Figure 1—figure supplement 25), which serves to minimize non-shape variations (size, location, and orientation) among specimens by rescaling, repositioning and rotating raw landmark configurations. After superimposition, centroid size of the palate for each specimen is calculated as square root of the sum of squared distances of all Procrustes landmarks of the palate from their corresponding centroid. The mean shape of specimen/species are calculated as the consensus from the superimposed landmark coordinates. Procrustes variances/distance between specimens are calculated as the square root of the sum of the squared distances between corresponding landmarks (Klingenberg, 2016). The mean value for each species (the species mean thereafter) in the composite cladogram was obtained by averaging Procrustes coordinates and centroid sizes when there are more than one representative specimens.

The geometry of the palate shows object symmetry (Mardia et al., 2000) with landmarks #15 and #20 lying in the midsagittal axis of the skull and the remaining 11 pair landmarks being generally symmetric about the mid-sagittal plane (Figure 1A). To calculate respective contributions to the total amount of shape variation from symmetric and asymmetric shape components, we used the function ‘C1v’ in the software R to create a new double 24-raw-landmark dataset that contains all original raw landmark coordinates and their mirrored copy reflected about the midsagittal axis and relabeled to match the original landmark numbers (Savriama, 2018). This doubled 24-raw-landmark dataset was then imported into MorphoJ for superimposition, covariation matrix buildup and PCA. Considering that the symmetric and asymmetric components of shape variations occupy complementary subspaces in the shape tangent space defined by the doubled datasets for landmark configurations with object symmetry (Mardia et al., 2000; Klingenberg et al., 2002), the relative amount of contributions to shape changes from symmetric and asymmetric components equals to the sum of the eigenvalues of their corresponding PCs derived from the PCA. This procedure does not apply to the seven-landmark dataset because the right vomer has no object symmetry.

Allometric analysis

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To verify if allometry, covariation of shape with size, plays a role in shape variations, we first performed a regular multivariate regression (Monteiro, 1999; Klingenberg, 2016) of the symmetric shape components (dependent variables) from the 24-Procrustes-landmark dataset on the log-transformed centroid size (log [CS]; independent variable) across all 70 specimens and the 34 species by using the ‘procD.lm’ function in the ‘geomorph’ R package (version 4.0.0; Adams et al., 2021; Collyer and Adams, 2021). Then we conducted a permutation test of 10,000 iterations using the same function to test the significance of these two regular regressions, respectively, considering that our sample size (n=70 for specimens; n=34 for species) is not considerably larger than the number of variables (n=49) for the 24-Procrustes-landmark dataset. Statistical results from these regular regression analyses may be inaccurate given that variations (or residuals) in the shape components among species are correlated with their evolutionary relationships (‘phylogenetic non-independence’ in Felsenstein, 1985). To account for impacts from the phylogeny, we conducted a phylogenetic regression (or evolutionary allometry analysis) of the shape components on log (CS) across species by using the function ‘procD.pgls’ of ‘geomorph,’ which is a distance-based phylogenetic generalized least squares method (Adams, 2014a) that uses our composite cladogram (without Aviturus) to remove phylogenetic covariances among species under a Brownian motion model of evolution.

To remove impacts from allometric shape components, shape variables (Procrustes-landmark coordinates in shape tangent space) were transformed by using residuals from the regular multivariate regression of shape on log (CS) across 70 specimens and 34 species, respectively, by using the function ‘procD.lm’ in ‘geomorph’ (Klingenberg, 2016). Non-allometric portion of the symmetric shape components are used for downstream statistical analyses.

Phylogenetic signal

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The phylogenetic signal is termed based on a pattern that closely related species tend to have similar character values stemmed from their shared evolutionary history (Felsenstein, 1985; Blomberg et al., 2003; Adams, 2014b). Whether the shape of the palate bears any phylogenetic signal was tested by using the Kmult method in the function ‘physignal’ of ‘geomorph,’ which uses the composite cladogram to account for phylogenetic non-independence under a Brownian motion model of evolutionary divergence, and permutates shape components of terminal taxa against the composite cladogram (without Aviturus). A phylogenetic signal analysis was also conducted for the right vomer (non-allometric seven-Procrustes-landmark dataset) using the composite cladogram including Aviturus. The resulting Kmult value for each of the two analyses measures the phylogenetic signal as a ratio of observed to expected shape variation obtained with and without considering phylogenetic non-independence (Adams and Collyer, 2019). When the Kmult value is larger than and equals to 1, closely related taxa resemble each other phenotypically more than expected under Brownian model, whereas when the value is smaller than 1, closely related taxa are less similar to one another phenotypically than expected under Brownian model (Adams, 2014b).

Morphological disparity and ecological indication of the palate

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Taxonomically, the five groups considered here are Pancryptobrancha, Hynobiidae, stem hynobiids, basal cryptobranchoids and non-cryptobranchoid early salamanders. The Paleocene Aviturus was classified into the clade Pancryptobrancha (Gubin, 1991; Vasilyan and Böhme, 2012). The Late Jurassic Beiyanerpeton represents a basal member of Salamandroidea (Gao et al., 2013) and the two stem urodeles Karaurus and Kokartus were classified into the family Karauridae (Skutschas and Martin, 2011). Following Jia et al., 2021a, we apply the name Hynobiidae to the crown group of Panhynobia, and we consider crown and stem taxa of Panhynobia separately. As mentioned above, living hynobiids are mainly metamorphosed because individuals typically go through a short period of development during which larval features (e.g. external gills, gill slits) are resorbed (e.g. Kuzmin and Thiesmeier, 2001; Kami, 2004; Fei et al., 2006; Poyarkov et al., 2012), and B. londongensis represents the single facultatively neotenic hynobiid species; living cryptobranchids are generally referred to as neotenic or partially metamorphosed as their individuals live in water in the adult stage and retain a few paedomorphic features (Deban and Wake, 2000), except that the life history strategy of Aviturus remains unknown (Vasilyan and Böhme, 2012; but see Skutschas et al., 2018). It is noteworthy that many fossil taxa investigated here (e.g. Chunerpeton) bear more apparent neotenic features (e.g. external gills, gill rakers) than living cryptobranchids; however, we did not differentiate subgroups within neotenic taxa due to our sampling scope. Living habitats of cryptobranchoids in the adult stage outside the breeding season have been broadly classified into three types, namely aquatic, semiaquatic, and terrestrial (Fei et al., 2006; Rong, 2018; Fei and Ye, 2016; AmphibiaWeb, 2021). In order to understand if factors like taxonomic affiliations at the genus and family level, life history strategy and living habitat would contribute to morphological disparities of the palate, we conducted phylogenetic Procrustes ANOVAs for the non-allometric symmetric shape components of the 24-Procrustes-landmark dataset using the function ‘procD.pgls’ of ‘geomorph.’ Pair-wise comparisons for morphological disparity (Procrustes variance) for the palate, vomer, parasphenoid (Supplementary file 1B) and each landmark point (Supplementary file 1C–E) across each of the aforementioned category were performed using ‘morphol.disparity’ in ‘geomorph.’

Principal component analyses

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To visualize the patterns of shape changes in the morphospace of palate and to investigate the mechanisms framing up the disparity patterns, we conducted four types of PCA by using the function ‘gm.prcomp’ in ‘geomorph’: a standard PCA, a phylogenetically aligned component analysis (PaCA; Collyer et al., 2020), a phylomorphospace analysis (PA; Rohlf, 2002), and a phylogenetic principal component analysis (Phylo-PCA; Revell, 2009). The standard PCA is based on a covariance matrix of the non-allometric symmetric shape components across all 70 specimens, and the resulting first few PCs (eigenvalues) reveal predominant trends in shape disparity patterns.

The other three PCA are based on the composite cladogram (without Aviturus) and a covariance matrix of the non-allometric symmetric shape components across all 34 species, with the ancestral PC values (eigenvalue) for internal nodes (Figure 1—figure supplement 26) estimated by using the Brownian motion model of evolution. The resulting first few PCs derived from the PA reveal predominant trends in shape disparity patterns with the phylogeny superimposed in the morphospace. On the other hand, the first PC derived from PaCA reveal shape disparity patterns mostly associated with phylogenetic signal, and the first few PCs from Phylo-PCA are phylogenetic independent and therefore reveal factors (e.g. ecological preferences) other than phylogenetic signal that account for disparity patterns of the palate (see Collyer et al., 2020). A same analysis strategy was conducted for the non-allometric seven-Procrustes-landmark dataset to address the paleobiology of Aviturus (Figure 2—figure supplement 5). Visualizations of the results were achieved by using the functions ‘plot.gm.prcomp,’ ‘make_ggplot’ in ‘geomorph,’ and ‘ggplot’ in the package ‘ggplot2’ (version 3.3.5; Wickham, 2016).

Covariates of the palate

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Configurations of VTR have long been identified as informative in the classification of Cryptobranchoidea (Estes, 1981; Zhao and Hu, 1984; Fei et al., 2006) and were recently argued to be ecologically informative (Jia et al., 2021b). To test if non-shape variations of the palate are reliable indicators in reconstructing paleoecology of early salamanders, we chose the following five continuous and two categorical covariates: length ratio of the vomer in the palate; length ratio of the parasphenoid in the palate; vomerine tooth number on a single vomer; width ratio of VTR in the vomer; width ratio of the outer and inner branch of VTR; and both the position (relative to the vomerine plate: anterior, middle, mid-posterior or posterior) and arrangement (oblique, transverse, curved) of VTR. Measurements of these continuous and categorical features were collected by using VG Studio Max and the Fiji platform (version 1.8.0_172; Schneider et al., 2012), with missing values for Aviturus, Kokartus, and Pangerpeton reconstructed by using the function ‘phylopars’ in ‘Rphylopars’ R package (version 0.3.2; Goolsby et al., 2021). Values and states for the five continuous covariates were visualized by boxplot in ‘ggplot2’ and were compared among different ecological groups by using Mann-Whitney U-test (Figure 3). Association of the two categorical covariates with ecology were investigated by contingency table and Cochran-Mantel-Haenszel test (Figure 3—figure supplement 1 and Supplementary file 1I).

Ancestral state reconstruction and evolutionary rate

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The ancestral shapes of the palate for several internal nodes including the respective last common ancestors of Hynobiidae, Panhynobia, Pancryptobrancha, Cryptobranchoidea, Urodela, and Caudata were estimated by using maximum likelihood method in the ‘anc.recon’ function in the ‘Rphylopars’ R package (version 0.3.2; Goolsby et al., 2021). The ancestral value of the five continuous covariates for the internal nodes was estimated by using ‘phylopars’ in the same package (Supplementary file 1J). On the other hand, the ancestral states of the two categorical covariates and life history strategy were estimated using the function ‘ace’ in ‘ape’, with their likelihood for each of the internal node depicted as a pie chart (Figure 3). Wire plots for the ancestral shape of the internal nodes with reference to the mean shape were created by using the functions ‘mshape’ and ‘plotRefToTarget’ in ‘geomorph’.

Evolutionary rates for each of the 24 landmarks collected on the palate, the vomer, the parasphenoid, and the five continuous covariates from the palate were calculated by using the function ‘compare.multi.evol.rates’ in ‘geomorph’. Then evolutionary rates of the palate, the vomer and the parasphenoid among species across groups in ecology, life history strategy and taxonomic affiliation were calculated and compared by using the function ‘compare.evol.rates’ in ‘geomorph’ (Supplementary file 1H).

Results derived from this study were exported from R and were illustrated and assembled in Adobe Photoshop CC. Source codes for R used in this study is available at GitHub (https://github.com/SalamanderGeomorph/Salamander_Palate, copy archived at swh:1:rev:8cbdd82025b0bf987bb6211239a2e7dc56c615d5, Salaman, 2022).

Data availability

All data needed to evaluate the conclusions are included in the manuscript and the Supplementary file 1. Details of specimens, CT parameters and raw landmark coordinates and centroid sizes are available in three CSV files in the online Dryad repository (https://doi.org/10.5061/dryad.c59zw3r8x). Source codes for R and SAS used in this study are available at GitHub (https://github.com/SalamanderGeomorph/Salamander_Palate, copy archived at swh:1:rev:8cbdd82025b0bf987bb6211239a2e7dc56c615d5).

The following data sets were generated
    1. Jia J
    2. Li G
    3. Gao K
    (2022) Dryad Digital Repository
    Specimen list and landmark coordinates for the palate of early salamanders.
    https://doi.org/10.5061/dryad.c59zw3r8x

References

  1. Book
    1. Deban SM
    2. Wake DB
    (2000)
    Aquatic feeding in salamanders
    In: Schwenk K, editors. Feeding: Form, Function and Evolution in Tetrapod Vertebrates. Academic. pp. 65–94.
  2. Book
    1. Estes R
    (1981)
    Encyclopedia of Paleoherpetology. Part 2, Gymnophiona, Caudata
    Gustav Fischer Verlag.
  3. Book
    1. Evans SE
    2. Milner AR
    (1994)
    Middle Jurassic microvertebrate assemblages from the British Isles
    In: Fraser NC, Sues HD, editors. In the Shadow of the Dinosaurs: Early Mesozoic Tetrapods. Cambridge University Press. pp. 303–321.
  4. Book
    1. Fei L
    2. Hu S
    3. Ye C
    4. Huang Y
    (2006)
    Fauna Sinica Amphibia
    Science.
  5. Book
    1. Fei L
    2. Ye C
    (2016)
    Amphibians of China
    Science.
    1. Gubin YM.
    (1991)
    Paleocene salamanders from Southern Mongolia
    Paleontologicheskii Zhurnal 33:96–106.
    1. Ivachnenko MF
    (1978)
    Urodelans from the Triassic and Jurassic of Soviet Central Asia
    Paleontologicheskii Zhurnal 12:362–368.
    1. Kami HG
    (2004)
    The biology of the Persian Mountain Salamander, Batrachuperus persicus (Amphibia, Caudata, Hynobiidae) in Golestan Province, Iran
    Asiatic Herpetological Research 10:182–190.
  6. Conference
    1. Kligman B
    2. Stocker M
    3. Marsh A
    4. Nesbitt S
    5. Parker W
    (2021)
    New Late Triassic stem-caecilian form southwestern North America strengthens evidence for lissamphibian monophyly, and illuminates the anatomical, functional, and geographic origins of living caecilians
    The Society of Vertebrate Paleontology (ed.): virtual meeting conference program, 81st Annual Meeting.
  7. Book
    1. Kuzmin SL
    2. Thiesmeier B
    (2001)
    Mountain Salamanders of the Genus Ranodon
    Pensoft Publishers.
  8. Software
    1. R Development Core Team
    (2021) R: A language and environment for statistical computing
    R Foundation for Statistical Computing, Vienna, Austria.
  9. Book
    1. Rohlf FJ
    (2002)
    Geometric morphometrics and phylogeny
    In: MacLeod N, editors. Morphology, Shape and Phylogeny. Taylor & Francis. pp. 175–193.
  10. Book
    1. Schoch RR
    2. Milner AR.
    (2014)
    Temnospondyli I. Part 3A2 of Sues H-D (ed.): Handbook of Paleoherpetology
    München: Dr. Friedrich Pfeil.
    1. Skutschas PP
    2. Krasnolutskii SA
    (2011)
    A new genus and species of basal salamanders from the Middle Jurassic of Western Siberia, Russia
    Proceedings of the Zoological Institute RAS 315:167–175.
    1. Slice D
    2. Bookstein F
    3. Marcus L
    4. Rohlf F
    (1996)
    Appendix I: a glossary for geometric morphometrics
    Nato Advanced Science Institutes Series A: Life Sciences 284:531–551.
  11. Book
    1. Trueb L
    (1993)
    Patterns of cranial diversity among the Lissamphibia
    In: Hanken J, Hall BK, editors. The Skull, Volume 2: Patterns of Structural and Systematic Diversity. The University of Chicago Press. pp. 255–343.
  12. Book
    1. Wake DB
    2. Deban SM
    (2000)
    Terrestrial feeding in salamanders
    In: Schwenk K, editors. Feeding: Form, Function and Evolution in Tetrapod Vertebrates. Academic, San Diego. pp. 95–116.
  13. Book
    1. Zhao E
    2. Hu Q
    (1984)
    Studies on Chinese Tailed Amphibians
    Sichuan Scientific and Technical Publishing House.

Decision letter

  1. Min Zhu
    Reviewing Editor; Institute of Vertebrate Paleontology and Paleoanthropology, Chinese Academy of Sciences, China
  2. George H Perry
    Senior Editor; Pennsylvania State University, United States
  3. Min Zhu
    Reviewer; Institute of Vertebrate Paleontology and Paleoanthropology, Chinese Academy of Sciences, China
  4. Pavel Skutschas
    Reviewer
  5. David Marjanovic
    Reviewer; Museum für Naturkunde, Germany

Our editorial process produces two outputs: i) public reviews designed to be posted alongside the preprint for the benefit of readers; ii) feedback on the manuscript for the authors, including requests for revisions, shown below. We also include an acceptance summary that explains what the editors found interesting or important about the work.

Decision letter after peer review:

Thank you for submitting your article "Palatal morphology predicts the paleobiology of early salamanders" for consideration by eLife. Your article has been reviewed by 3 peer reviewers, including Min Zhu as the Reviewing Editor and Reviewer #1, and the evaluation has been overseen by a Reviewing Editor and George Perry as the Senior Editor. The following individuals involved in the review of your submission have agreed to reveal their identity: Pavel Skutschas (Reviewer #2); David Marjanovic (Reviewer #3).

The reviewers have discussed their reviews with one another, and the Reviewing Editor has drafted this to help you prepare a revised submission.

Essential revisions:

Using geometric morphometric analysis, you demonstrate that both the shape of the palate and several non-shape variables (particularly associated with vomerine teeth) are ecologically informative in early stem- and basal crown-group salamanders. If the used phylogenetic tree is accurate, their conclusions are robust. Please discuss what pitfalls might have been encountered in constructing the tree. Please better summarize the innovative aspects of the work and pay attention to the minor points raised by the reviewers.

Reviewer #1 (Recommendations for the authors):

The manuscript in the present format has the obvious weakness in the summarization of innovative points and needs revisions and more stylistic works.

Title: This looks like an overstatement of the present research.

Abstract: More words on the background, and clear summarization of innovative points. The logic connection between the present points is loose.

Line 21: "phylotypic designs" is not suitable for evolution.

Line 29: The sentence needs re-written. 'living … representatives from the Triassic'?

Line 47: 'evolutionary history of paleoecology'?

Lines 50-51: "crown + stem" replaced by "total group".

Line 52: "as sister-group taxa" is redundant here.

Lines 65-66: What is the logic here? How about the record of stem urodeles? Early salamanders (Line 42) should include stem urodeles.

Line 82: Is Triassurus a stem urodele? To be mentioned here.

Line 103: 'basal' replaced by 'fossil' to parallel with 'living'.

Line 113: add 'a' before 'stepwise pattern'.

Lines 114-118: Redundant with the contents in the concluding paragraph of the main text. To be deleted.

Line 133: 95.15% among 70 specimens? To be clarified here.

Lines 240 and other places. What is the difference between 'variables' and 'covariates'?

Line 241: 'most of the five' represents how many? Delete 'most of'?

Line 243: branches.

Lines 298-299. Meaningless. What do you mean by 'unhelpful'?

Line 303: To be clarified.

Line 309: Replaced by 'paleoecological turnover.

Line 314: Replaced by 'resulting'.

Line 320: 'other' to be deleted?

Line 339: 'fossil' is redundant here.

Line 360: water current?

Line 428: source data for rate?

Lines 435-438: The concluding sentence should be re-formatted.

Lines 480, 484: check words 'partes', 'pas'.

Reviewer #3 (Recommendations for the authors):

This manuscript is a valuable contribution to evolutionary ecomorphology in extant and extinct tetrapods. I recommend publication in eLife after appropriate revision.

The conversion of the manuscript to PDF format has caused a few problems. The text has suffered from encoding issues: some colons and probably all dashes have been replaced by squares, and seemingly random parts of the text have been replaced by randomly selected all-caps letters superimposed with squares, or by squares and a lot of white space. As a consequence, there are parts of the manuscript I cannot evaluate because, in extreme cases, entire lines are missing. Please fix this problem before the next round of review. The lines that this concerns are 37, 69, 75, 100, 123, 143, 153, 157, 187, 193, 202, 211, 216, 221, 257, 276, 474, 503, 514-516 (these three lines are almost completely obliterated), 519, 533, 541, 556, 573, 574, 581, 583, 584, 590, 597, 598, 635, 636, 639, 643, 651, 659, 661, 676, 677, 680, 686, 688, 690, 693, 696, 699, 726, 728, 764, every page range in the references, 825, 826, 904, 905, 932, 967, 970, 980, 973, and the name of every figure supplement.

In a few places the writing is difficult to parse, slowing readers down unnecessarily.

The lack of sirenids and salamandroids elegantly avoids the problem of the phylogeny of early salamanders (see below), but it means that crown-group salamanders are represented only by cryptobranchoids and maybe Beiyanerpeton. This greatly restricts this study's ability to reconstruct the first crown-group salamander. Given that sirenids and salamandroids are sister-groups and that all known sirenids (Cretaceous to extant) are only partially metamorphosed (much like Andrias), it is possible that adding them (and a few unquestioned salamandroids) to the datasets would modify the conclusions. This should either be tested – which would require repeating all your phylogeny-informed analyses, ideally twice to account for different phylogenetic hypotheses – or the conclusions about the first crown-group salamander should be strongly deemphasized in the text.

Following earlier analyses by the first and the last author, the phylogenetic positions of all mentioned Mesozoic salamanders are simply stated as facts. I'm surprised the work of Rong et al. (2020) is nowhere cited. It showed that the existing datasets for phylogenetic analysis of early salamanders are riddled with too many inaccuracies and redundant characters to be reliable. While Rong et al. (2020) did not undertake the necessary complete review of these datasets, which means their conclusions are not wholly reliable either, they did show that modest improvements result in cladograms that show Chunerpeton (redescribed in that paper), Beiyanerpeton, Qinglongtriton and possibly all other Mesozoic Chinese salamanders outside the salamander crown group. The reconstruction not only of the first crown-group salamander, but also of the first crown-group cryptobranchoid is affected.

Rong et al. (2020) further pointed out the nomenclatural fact that "herpeton" is grammatically neuter and that therefore the International Code of Zoological Nomenclature automatically corrects a number of species names from "-is" (masculine or feminine) to "-e" (neuter). The authors and dates of the names are not affected by this.

You follow common usage among paleontologists since the early 1990s in calling the crown group of salamanders Urodela and the total group Caudata. Apparently without talking to anyone, Wake (2020) has defined the name Caudata as applying to the crown group of salamanders in a way that is valid under the International Code of Phylogenetic Nomenclature (Cantino and de Queiroz, 2020). Wake (2020) explicitly left the name Urodela undefined. It might be best if you mention this situation in a few words in the manuscript. In the longer run, beyond this manuscript, it would probably be best to ask the Committee on Phylogenetic Nomenclature for an emendation of the definition of Caudata. I'm a member of the Committee and would happily coauthor a paper for this purpose with you and other experts on salamanders.

Lines 30, 40: I don't think Chinlestegophis and Rileymillerus should be accepted as undoubted caecilians. But as it happens, an undoubted Late Triassic caecilian was recently announced in a published conference abstract by Kligman et al. (2021), so the oldest known caecilians are Late Triassic in age either way.

35: These two papers are neither the most recent nor in any other sense the most important ones on this subject. I recommend citing Pardo et al. (2017b) and Daza et al. (2020: Figure 4D, E, S13, S14) instead.

36: "2017b" is an error for "2017a".

37: The review paper by myself and Laurin (2013) is not up to date, being written long before completion of the large phylogenetic analysis by myself and Laurin (2019), let alone its update by Daza et al. (2020: Figure 4F, S15), not to mention the analysis of ontogeny by Laurin et al. (2022). Importantly, it is clear that Lissamphibia is not derived from aquatic lepospondyls.

40-42: This is plainly not true. Chinlestegophis obviously lived in burrows, but apart from its elongate body shape it shows no adaptations to burrowing. Even the Early Jurassic Eocaecilia and, as far as its fragmentary remains allow us to tell, the Early Cretaceous Rubricacaecilia lack some of the crown group's adaptations to burrowing, e.g. they retain limbs, larger orbits and more sutures in the skull.

49: Of any two sister groups, each is more primitive than the other in some respects but not in others. It makes little sense to call Cryptobranchoidea "the most primitive clade of the crown group salamanders". I suggest "the sister group to all other crown salamanders".

57, 625-626: For the life history of Aviturus, see Skutschas et al. (2018).

58-59: It is not excluded that Regalerpeton is a stem-group salamander (Rong et al. 2020: Figure 5).

71, 73: Two more good opportunities to cite Rong et al. (2020).

89, 173-179, 446-450, 615: but see Rong et al. (2020) for reasons for skepticism.

110, 198, 268, 271, 276, 277, 279, 280, 282, 284, 286, 289, 290, 334, 337, 428: Insert "last" before "common"!

194: Replace "albert" by "albeit" (or "although" or just "though").

202, 602, 635: Uppercase for Procrustes (the name of a mythological person).

293-294: All adult anurans and caecilians are metamorphosed, but some of both are fully aquatic (e.g. Pipidae, Typhlonectidae). I would therefore rearrange the sentence to: "Among modern amphibians, the adults of anurans and caecilians are metamorphosed, and most of them are terrestrial."

299: …or whatever will remain of Lepospondyli.

308: I would write "metamorphic taxa" or "metamorphosed individuals".

339-340: Aviturus, which dates from the very end of the Paleocene, is not the earliest known pancryptobranchan. The earliest entirely undoubted one is "Cryptobranchus" saskatchewanensis, which is a few million years older (late middle Paleocene). There is evidence that the much older (mid-Cretaceous) Eoscapherpeton is a stem-pancryptobranchan; see Marjanović (2021: supplementary material pp. 13-16) for references and a brief review.

480: The plural of pars palatina is partes palatinae – as in most languages of Europe (but unlike English), number and gender (and case) are marked on adjectives as well as nouns in Latin.

484: Replace "pas" by "pars".

791: Replace "batrachian" by "batrachians".

857-859: Unlike English, German does not have separate capitalization rules for headlines – but it always gives a capital Letter to every Noun, while almost nothing else is ever capitalized, not even Adjectives derived from proper Names. Therefore, please correct the Title to: "Generelle Morphologie der Organismen: Allgemeine Grundzüge der organischen Formen-Wissenschaft, mechanisch begründet durch die von Charles Darwin reformirte Descendenz-Theorie".

Finally, the supplementary file claims to be a PDF file ("This PDF file contains supplementary file 1A to 1K."), but it is a DOCX file. In the interest of wider accessibility, I recommend you convert it to PDF before resubmitting it.

I hope these comments are helpful. I apologize again for the delay, and I'm looking forward to the next version of your manuscript!

References not cited in the manuscript:

Cantino PD, de Queiroz K. 2020. International Code of Phylogenetic Nomenclature (PhyloCode). Version 6. CRC/Taylor & Francis/Informa, Boca Raton/London/New York. ISBN: 978-1-138-33282-9 (paperback), 978-1-138-33286-7 (hardback), 9780429446320 (e-book). DOI: https://doi.org/10.1201/9780429446320 Openly accessible at http://phylonames.org/code/

Daza JD, Stanley EL, Bolet A, Bauer AM, Arias JS, Čerňanský A, Bevitt JJ, Wagner P, Evans SE. 2020. Enigmatic amphibians in mid-Cretaceous amber were chameleon-like ballistic feeders. Science 370:687-691. DOI: https://doi.org/10.1126/science.abb6005

Kligman B, Stocker M, March A, Nesbitt S, Parker W. 2021. New Late Triassic stem-caecilian from southwestern North America strengthens evidence for lissamphibian monophyly, and illuminates the anatomical, functional and geographic origins of living caecilians [abstract]. Society of Vertebrate Paleontology (ed.): Virtual meeting conference program, 81st annual meeting, p. 160. The entire abstract volume can be downloaded here: https://vertpaleo.org/svp_2021_virtualbook_final/

Laurin M, Lapauze O, Marjanović D. 2022. What do ossification sequences tell us about the origin of extant amphibians? Peer Community Journal 2:e12. DOI: https://doi.org/10.24072/pcjournal.89

Marjanović D. 2021. The making of calibration sausage exemplified by recalibrating the transcriptomic timetree of jawed vertebrates. Frontiers in Genetics 12:521693. DOI: 10.3389/fgene.2021.521693

Marjanović D, Laurin M. 2019. Phylogeny of Paleozoic limbed vertebrates reassessed through revision and expansion of the largest published relevant data matrix. PeerJ 6:e5565. DOI: https://doi.org/10.7717/peerj.5565

Rong Y-F, Vasilyan D, Dong L-P, Wang Y. 2020 (printed 2021). Revision of Chunerpeton tianyiense (Lissamphibia, Caudata): Is it a cryptobranchid salamander? Palaeoworld 30:708-723. DOI: https://doi.org/10.1016/j.palwor.2020.12.001

Skutschas PP, Kolchanov VV, Bulanov VV, Sennikov AG, Boitsova EA, Gulbev VK, Syromyatnikova EV. 2018 (printed 2020). Reconstruction of the life history traits in the giant salamander Aviturus exsecratus (Caudata, Cryptobranchidae) from the Paleocene of Mongolia using zygapophyseal skeletochronology. Historical Biology 32:645-648. DOI: https://doi.org/10.1080/08912963.2018.1523157

Wake DB. 2020. Caudata J. A. Scopoli 1777 [D. Wake], converted clade name. [Brackets in the original.] de Queiroz K, Cantino PD, Bauthier JA (eds): Phylonyms. A Companion to the PhyloCode (CRC/Taylor & Francis/Informa, Boca Raton/London/New York), pp. 785-787. ISBN: 978-1-138-33293-5 (hardback), 9780429446276 (e-book). DOI of the entire book: https://doi.org/10.1201/9780429446276

[Editors' note: further revisions were suggested prior to acceptance, as described below.]

Thank you for resubmitting your work entitled "Palatal morphology predicts the paleobiology of early salamanders" for further consideration by eLife. Your revised article has been evaluated by George Perry (Senior Editor) and a Reviewing Editor.

The manuscript has been improved but there are some remaining issues that need to be addressed, as outlined below:

Reviewer #1:

They have addressed my concerns in my previous review and revised their manuscript accordingly. I have no additional recommendations for the authors.

Reviewer #3:

The issues with image quality in the merged PDF have disappeared. Please correct the species names in figures 1, 3, 19, 23-26, 30-32, 36 and 38 if I've counted correctly, and replace "semiaqaic" with "semiaquatic" in Figure 3.

Sirenidae and the first crown-group salamander

Your anatomical reasons for the omission of sirenids are convincing, and I had indeed confused sirenids and amphiumids in my statement about partial metamorphosis – thank you for pointing this out! I am, however, quite surprised that you present the phylogenetic position of Sirenidae as a mystery. Sirenidae and Salamandroidea have been found as sister groups in every large-scale study of molecular data, no matter which data or which method exactly: 7,189 transcripts of nuclear genes and Bayesian inference (Irisarri et al. 2017; 99% posterior probability), 5 mitochondrial and 10 nuclear genes and maximum likelihood (Vijayakumar et al. 2019: supplementary file Amphibia_New_India_SHL_Dryad.tre; 52% bootstrap support – note this is the latest and largest version of R. A. Pyron's series of matrices, and all versions, including Jetz and Pyron [2018] which you cite, found the same topology), or 120 nuclear protein-coding genes, separately and together, and maximum likelihood (Himes et al. 2020: 100% bootstrap and ASTRAL posterior values). Given such extraordinary agreement, the fact that the morphological evidence is less clear can be blamed on the morphological evidence, or on our lack of knowledge of it (I greatly appreciate your recent work in describing the osteology of extant salamanders!), but it would be very difficult to try to argue the molecular data away. Therefore, I think you should mention in the text that you cannot include Sirenidae in your dataset for the reasons you presented in your response and that this is a potential weak point in inferences about the last common ancestor of all crown-group salamanders.

You point out that the analyses of Rong et al. (2020, printed 2021) failed to find Hynobiidae or Cryptobranchoidea. Unfortunately, this is not incompatible with their matrix being an improvement over its sources. It is not uncommon (e.g. Marjanović and Laurin, 2019, and references therein) for improvements to matrices for phylogenetic analysis to decrease resolution, and it is not uncommon either for matrices to support the right things for the wrong reasons. Matrices that support clades for the wrong reasons are unreliable in the sense that they cannot be trusted to place added taxa accurately. As I stated, the version by Rong et al. (2020) cannot be relied upon as strong evidence for the phylogenetic positions of Chunerpeton, Beiyanerpeton or any other taxon – but that does not make the earlier versions of that matrix any better. They contain, after all, such phenomena as redundant and even duplicate characters: for example, the haploid and the diploid number of chromosomes are both included as separate characters in the previous versions. Duplicating a character inevitably distorts, if not the topology, then at the very least the support values for that topology (references in Marjanović and Laurin, 2019: 15-16). Rong et al. (2020) made a certain effort to reduce this problem.

They also ordered certain characters, unlike their sources. Ordering is widely believed to be a philosophical question, but there is strong evidence from simulations as well as from empirical studies that potentially clinal or meristic characters must be ordered to avoid inaccurate results as well as both false negatives and false positives in resolution (references and brief discussion in Marjanović and Laurin, 2019: 16).

Phylogenetics with morphological data

Parsimony analysis of morphological data should not be treated as a black box.

Daza et al. (2020) did all their phylogenetic work on previously published matrices; they added or updated the scores of Albanerpetidae, but did not make any other changes to those matrices. Therefore, practically all the problems with their results are the fault of those matrices. The clearly wrong placement of Chelotriton in their Figure 4F and S15 stem from the fact that I had added Chelotriton to the source matrix in question, that of Marjanović and Laurin (2019), which is simply not equipped to handle crown-group salamanders. (That matrix is an attempt to improve the one by Ruta and Coates, 2007. In order to limit the amount of time and effort, we did not add any characters to that matrix. The matrix of Ruta and Coates, 2007, contained only two salamanders – Karaurus and Valdotriton – and therefore lacked any characters specific to salamander phylogeny.) I did this in order to test a point on ontogeny and phylogeny: would the extreme metamorphosis of Chelotriton, which makes it look more like a temnospondyl than lissamphibians usually do, pull some or all lissamphibians into Temnospondyli? That did not happen, but Chelotriton was pulled out of the salamander clade. The latter result is obviously wrong, and obviously due to the lack of salamander-specific characters in the matrix; there is no reason to think that it indicates a more general problem. The former result, on the other hand, is actually strengthened that way.

In another analysis, Daza et al. (2020: Figure 4E, S14) found Karauridae outside Batrachia. I haven't looked into its precise causes, but it is simply shared with the source matrix of that analysis, the matrix of Pardo et al. (2017a).

Both of these analyses are, however, relevant to the ecology of the first lissamphibian by finding Albanerpetidae as the sister group of Lissamphibia, Albanerpetidae being terrestrial and fully metamorphic or of course direct-developing (early juveniles of any sort are not known). This is a data point for phylogenetic bracketing.

In the context of the specimens referred to Chunerpeton by Rong et al. (2020), you write: "only those morphological features in adults are trustworthy for taxonomic and phylogenetic interpretations." In phylogenetics, however, morphological features are not automatically comparable just because they are found in sexually mature adults, as you seem to have assumed in your published phylogenetic work: for much of the skeleton, for instance, neotenic adults are much more easily comparable to larvae than to adults of metamorphic taxa, so that the presence or absence of many character states of metamorphic adults must be scored as unknown/inapplicable in neotenic taxa. This has a strong effect on the topologies found by phylogenetic analyses (Wiens et al., 2005; Marjanović and Laurin, 2019: 21-22, and references therein).

Nomenclature and taxonomy

I greatly appreciate your comments on the validity of the referral of the new specimens by Rong et al. (2020) to Chunerpeton, and I'm looking forward to your further publications on this matter!

Likewise, I appreciate your comments on the definitions of the names Caudata and Urodela and will try to send you a draft manuscript as soon as possible. For the purposes of the present manuscript, I withdraw all my objections.

Adaptations of Chinlestegophis

Thank you for reminding me of the features listed as adaptations to burrowing by Pardo et al. (2017a). I agree that the fairly small orbits may count as such, though they could also hint at life at the bottom of muddy bodies of water – a strictly aquatic lifestyle, apart from sheltering in burrows with 100% humidity, is assured by the lateral-line grooves. The position of the jaw articulation, however, is shared with all other brachiopods, where it is an adaptation to a particular style of suction feeding; it is in fact more extreme in Batrachosuchus, depicted in Pardo et al. (2017a: Figure 3). By "consolidation of the skull", Pardo et al. (2017a: supp. inf. part F) mean the frankly irrelevant fusion of lacrimal and maxilla – besides, it is not in fact clear whether a lacrimal is even present -, the supposed fusion of the pterygoid and the quadrate for which there is very little evidence presented in the paper, and fusion of the exoccipitals and the basioccipital which is universal in dissorophoid temnospondyls, none of which were burrowing or had any sort of digging lifestyle – and note that it is not clear if a basioccipital was present in the first place; it remained cartilaginous and very small in other brachiopods. In a borrower, one would expect, as in extant caecilians, a well-ossified braincase that could function as a strut to enable the skull to resist rostrocaudal compression, dorsoventral bending, or twisting; yet, most of the braincase is not ossified at all in Chinlestegophis (Pardo et al. 2017: supp. inf. part B). This is particularly striking in comparison to the burrowing "lepospondyls" described by Pardo and various coauthors in the two years prior.

Interestingly, the stem-caecilian presented by Kligman et al. (2021) seems not to be adapted to burrowing at all, but it is clearly much closer to the crown group than Chinlestegophis.

Details

Lines 38-41: I had not quite appreciated that here you cite references both for the lifestyles and the phylogenetic positions of the listed taxa, and only suggested references for the phylogenetic positions of various "lepospondyls". For the terrestrial lifestyle of at least one amphibamid, I recommend Laurin et al. (2004) and references therein. For the various lifestyles of "lepospondyls", I recommend Jansen & Marjanović (2021) and references therein. While some stereospondyls have occasionally been considered semiaquatic for vaguely articulated reasons, almost all were certainly fully aquatic as shown by the lateral-line grooves on their skulls and further supported by their very slow peri- and endochondral ossification; I recommend the brief but clear statement in Schoch & Milner (2014: 123) and references therein. Notably, Chinlestegophis has lateral-line grooves (Pardo et al., 2017a), showing that it was not semiaquatic; "reduced" grooves as identified by Pardo et al. (2017a) mean that most of the lateral-line organ was situated in the skin and did not contact the bone, not that the organ was "reduced" – something that does not occur, because the lateral-line organ dries up and dies from serious exposure to air as inevitably caused by a semiaquatic lifestyle. – While Jansen & Marjanović (2021) is published on a preprint server with the usual disclaimer, it is in fact an accepted manuscript published with permission from the journal (Comptes Rendus Palevol) that accepted it after peer review. The journal is currently changing publishers, a process that started before the pandemic and is still not complete; we've been waiting for the page proofs since March 2021.

80-81: Other than its probable membership in Karauridae, is there evidence on the lifestyle of Marmorerpeton?

85, 106: If you use Caudata as the name for the total group, Triassurus is a stem caudate, not a stem urodele; nothing is a stem urodele, because if something is on the stem from which Urodela comes, it is outside Urodela by definition. At least for the first few decades of this terminology, a clade consisted of a crown group and a stem-group; to be a stem urodele, something has to be a urodele.

106: Likewise, if you use Urodela as the name for the crown group, "crown urodeles" is redundant; "urodeles" would be enough.

623-624: I would rather write: "As implied by Jia et al. (2021a), we apply the name Hynobiidae to the crown group of Panhynobia".

912: "lepospondyl", not "lepospondyls".

928: "Kuro-o", a Japanese name with three syllables; "oo" could be misunderstood as a long vowel – Japanese distinguishes long from short vowels. Sometimes apostrophes are used to disambiguate in transcriptions, sometimes hyphens are used instead.

976: "Syromyatnikova".

977: italics for the genus & species name (present in the original, I've checked)

Suggestions on style and language

Lines 15-18: "but the small number of reliable ecological indicators established so far hinders investigations into the paleobiology of early salamanders. Here we statistically demonstrate, by using time-calibrated phylogenetic trees and geometric morphometric analysis on 71 specimens in 36 species, that both the shape"…

21: I'm not sure what you mean by "strictly". Perhaps "analyzed in detail" would be clearer?

25-27: That would mean the disparities, not the salamanders, have achieved the ecological preferences. Would the following be an improvement? "Salamanders began to diversify ecologically before the Middle Jurassic and achieved all their present modes of life in the Early Cretaceous."

References cited above but not in the manuscript

Hime PM, Lemmon AR, Moriarty Lemmon EC, Prendini E, Brown JM, Thomson RC, Kratovil JD, Noonan BP, Pyron RA, Peloso PLV, Kortyna ML, Keogh, JS, Donnellan SC, Lockridge Mueller R, Raxworthy CR, Kunte K, Ron SR, Das S, Gaitonde N, Green DM, Labisko J, Che J, Weisrock DW. 2020. Phylogenomics reveals ancient gene tree discordance in the amphibian tree of life. Systematic Biology 70:49-66. DOI: 10.1093/sysbio/syaa034

Laurin M, Girondot M, Loth M-M. 2004. The evolution of long bone microstructure and lifestyle in lissamphibians. Paleobiology 30:589-613. DOI: 10.1666/0094-8373(2004)030<0589:TEOLBM>2.0.CO;2

Irisarri I, Baurain D, Brinkmann H, Delsuc F, Sire J-Y, Kupfer A, Petersen J, Jarek M, Meyer A, Vences M, Philippe H. 2017. Phylotranscriptomic consolidation of the jawed vertebrate timetree. Nature Ecology & Evolution 1:1370-1378. DOI: 10.1038/s41559-017-0240-5

Jansen M, Marjanović D. 2021. The scratch-digging lifestyle of the Permian "microsaur" Batropetes as a model for the exaptative origin of jumping locomotion in frogs. bioRχiv 460658. DOI: 10.1101/2021.09.27.460658

Schoch RR, Milner AR. 2014. Temnospondyli I. Part 3A2 of Sues H-D (ed.): Handbook of Paleoherpetology. München: Dr. Friedrich Pfeil.

Vijayakumar SP, Pyron RA, Dinesh KP, Torsekar VR, Srikanthan AN, Swamy P, Stanley EL, Blackburn DC, Shanker K. 2019. A new ancient lineage of frog (Anura: Nyctibatrachidae: Astrobatrachinae subfam. nov.) endemic to the Western Ghats of Peninsular India. PeerJ 7:e6457. DOI: 10.7717/peerj.6457

https://doi.org/10.7554/eLife.76864.sa1

Author response

Essential revisions:

Using geometric morphometric analysis, you demonstrate that both the shape of the palate and several non-shape variables (particularly associated with vomerine teeth) are ecologically informative in early stem- and basal crown-group salamanders. If the used phylogenetic tree is accurate, their conclusions are robust. Please discuss what pitfalls might have been encountered in constructing the tree. Please better summarize the innovative aspects of the work and pay attention to the minor points raised by the reviewers.

Reviewer #1 (Recommendations for the authors):

The manuscript in the present format has the obvious weakness in the summarization of innovative points and needs revisions and more stylistic works.

Title: This looks like an overstatement of the present research.

We pondered this comment and we firmly believe this title does justice to the aim, analyzes and conclusions of our manuscript. We believe that the top priority to understand the evolutionary paleobiology of early salamanders in this study is to establish osteological indicators for ecology in living taxa, which are the shape of the palate and seven covariates associated with the vomer and vomerine teeth, and demonstrate that these indicators are reliable through a set of strict statistical analyses. Then these ecological indicators are applied to fossil taxa to recover their paleoecological preference and reconstruct the ancestral configurations of these indicators in the hypothesized ancestors of different salamander clades.

Abstract: More words on the background, and clear summarization of innovative points. The logic connection between the present points is loose.

Please see the revised Abstract section in the resubmitted manuscript. We appreciate this advice.

Line 21: "phylotypic designs" is not suitable for evolution.

To avoid confusions, “phenotypic designs” is now replaced by “phenotypic configurations” in Line 21 and Line 378.

Line 29: The sentence needs re-written. 'living … representatives from the Triassic'?

The sentence “Salamanders, anurans and caecilians are highly distinctive from one another in their morphology in both living and geologically the oldest known representatives from the Triassic” is now re-written as “Salamanders, anurans and caecilians are highly distinctive from one another in their morphology in both living species and their respective oldest known relatives from the Triassic”.

Line 47: 'evolutionary history of paleoecology'?

“evolutionary history of paleoecology” changed to “evolutionary paleoecology”.

Lines 50-51: "crown + stem" replaced by "total group".

done.

Line 52: "as sister-group taxa" is redundant here.

“as sister-group taxa” is deleted.

Lines 65-66: What is the logic here? How about the record of stem urodeles? Early salamanders (Line 42) should include stem urodeles.

Right, early salamanders do include both stem and basal crown urodeles. We did originally include introductions on stem urodeles in the posterior half of this paragraph, starting from the sentence----“In contrast, other contemporaries (e.g., Kokartus, Marmorerpeton) from the Middle Jurassic Bathonian of UK, Russia and Kyrgyzstan are all neotenic and aquatic at their adult stage, and have been classified as stem urodeles by…”

We reworded the first sentence of this paragraph to better emphasize the importance of Cryptobranchoidea in understanding the early paleoecology of salamanders, and now it goes like this----“Cryptobranchoidea are critical in understanding the paleoecology of early salamanders because the earliest known cryptobranchoids from the Middle Jurassic Bathonian have higher disparities in both life history strategies and ecological preferences than stem urodeles, and represent the oldest known crown urodeles, including ‘Kirtlington salamander B’ from the UK,….”.

Line 82: Is Triassurus a stem urodele? To be mentioned here.

It is a stem urodele. “The only known pre-Jurassic salamander, Triassurus…” is now replaced by “The only known pre-Jurassic stem urodele, Triassurus…”.

Line 103: 'basal' replaced by 'fossil' to parallel with 'living'.

We replaced the first “basal” in “basal genera of cryptobranchoids” by “fossil”, and kept the second “basal” in “stem and basal crown urodeles” to parallel with “stem”.

Line 113: add 'a' before 'stepwise pattern'.

done.

Lines 114-118: Redundant with the contents in the concluding paragraph of the main text. To be deleted.

The two sentences at Lines 114-118 are now deleted.

Line 133: 95.15% among 70 specimens? To be clarified here.

Correct. We added “in 70 specimens” after “96.15% of the total shape variation”.

Lines 240 and other places. What is the difference between 'variables' and 'covariates'?

In statistics, both “variables” and “covariates” refer to characteristics of the participants in an experiment. Both can be used to refer independent variables, whereas covariates sometimes would have another layer of meaning when referring to the interrelationships among variables. We use “covariates” for the five continuous and two categorial non-shape variables and to differentiate them from the shape variables, which are 2D landmark points represented by x and y variables. We think “covariate” is accurate for non-shape variables because these seven covariates are mostly derived from the same subset of the palate (vomer + parasphenoid), the vomer/vomerine teeth, and any changes in the configuration of vomer/vomerine teeth can affect most of these covariates. So theses covariates are sort of “inter-related”. To be consistent, we replaced “variables” with “covariates” for non-shape variables in the manuscript.

Line 241: 'most of the five' represents how many? Delete 'most of'?

Only the first four out of the five continuous covariates listed within the parentheses are significantly impacted by allometry, not the fifth covariate (vomerine tooth number), and now we replaced “most” by “the first four”.

Line 243: branches.

done.

Lines 298-299. Meaningless. What do you mean by 'unhelpful'?

The earliest lissamphibians should have a unified lifestyle if they have a monophyletic origin. “unhelpful” here refers to the conflict between this hypothesized unified lifestyle in the earliest lissamphibians and the diverse lifestyles in modern lissamphibians. We now revised the sentence as the following: “The discrepancy in ecological preference between salamanders and anurans and caecilians is unhelpful in understanding the evolutionary paleoecology in the early lissamphibians given that they have a monophyletic origin from Temnospondyli.”

Line 303: To be clarified.

“…which to some extent are contributed by the lack of taphonomic analyses (Wang et al., 2019)” is now changed to “…which to some extent are contributed by the insufficient taphonomic analyses on fossil sites with salamander discoveries, such as the Daohugou fossil locality (Wang et al., 2019)”.

Line 309: Replaced by 'paleoecological turnover.

done.

Line 314: Replaced by 'resulting'.

replaced as suggested.

Line 320: 'other' to be deleted?

done.

Line 339: 'fossil' is redundant here.

“fossil” is removed.

Line 360: water current?

“a current of water” is replaced with “water current”

Line 428: source data for rate?

We added “(Supplementary file 1H)” after “…have the highest evolutionary rate in the palate” to show the source data for this statement.

Lines 435-438: The concluding sentence should be re-formatted.

We now have reformatted the concluding sentence as below: “Our results rigorously show that the shape of the palate and many non-shape covariates particularly associated with vomerine teeth are reliable ecological indicators for paleoecology of early salamanders, and we demonstrate that metamorphosis with the biphasic ecological preference (aquatic larvae + terrestrial adults) is not only the ancestral lifestyle in salamanders but also significant for the rise and diversification of modern amphibians.”

Lines 480, 484: check words 'partes', 'pas'.

To match our use of “paired vomers and a single median parasphenoid” in the same sentence, we chose to use the plural form of pars palatina for “premaxilla and maxilla”. The word “pars” is a singular noun in Latin (means part in English) and “palatina” is a singular nominative declining adjective in Latin (means palatine in English), and we agree with Dave (Reviewer #3) on that Latin adjectives should follow nouns in number, gender as well as case. The plural form for “pars palatina” therefore should be “partes palatinae” and thus we corrected our mistake. However, it is confusing to find both “partes palatina” and “partes palatinae” in published studies found through Google Scholar.

We also corrected our typo “pas” and replaced it with “pars” at Line 485.

Reviewer #3 (Recommendations for the authors):

This manuscript is a valuable contribution to evolutionary ecomorphology in extant and extinct tetrapods. I recommend publication in eLife after appropriate revision.

We thank you for your encouragement and please find our revisions in the re-submitted manuscript and our responses to your questions below.

The conversion of the manuscript to PDF format has caused a few problems. The text has suffered from encoding issues: some colons and probably all dashes have been replaced by squares, and seemingly random parts of the text have been replaced by randomly selected all-caps letters superimposed with squares, or by squares and a lot of white space. As a consequence, there are parts of the manuscript I cannot evaluate because, in extreme cases, entire lines are missing. Please fix this problem before the next round of review. The lines that this concerns are 37, 69, 75, 100, 123, 143, 153, 157, 187, 193, 202, 211, 216, 221, 257, 276, 474, 503, 514-516 (these three lines are almost completely obliterated), 519, 533, 541, 556, 573, 574, 581, 583, 584, 590, 597, 598, 635, 636, 639, 643, 651, 659, 661, 676, 677, 680, 686, 688, 690, 693, 696, 699, 726, 728, 764, every page range in the references, 825, 826, 904, 905, 932, 967, 970, 980, 973, and the name of every figure supplement.

In a few places the writing is difficult to parse, slowing readers down unnecessarily.

We apologize for any inconvenience caused by the flawed PDF and thank you for pointing out the places with words loss and format chaos. We believe the merged PDF is not the one we originally approved (48 MB in size) and instead is likely a size-compressed copy (~1.46 MB in size) uploaded to the Biorxiv Preprint platform (version 1). However, the Word file of our manuscript that we originally submitted works well though, and we also immediately uploaded a new PDF (as version 2; https://www.biorxiv.org/content/10.1101/2022.01.17.476642v2) to Biorxiv on January 22, 2022 when we noticed the original PDF was not working well.

The lack of sirenids and salamandroids elegantly avoids the problem of the phylogeny of early salamanders (see below), but it means that crown-group salamanders are represented only by cryptobranchoids and maybe Beiyanerpeton. This greatly restricts this study's ability to reconstruct the first crown-group salamander. Given that sirenids and salamandroids are sister-groups and that all known sirenids (Cretaceous to extant) are only partially metamorphosed (much like Andrias), it is possible that adding them (and a few unquestioned salamandroids) to the datasets would modify the conclusions. This should either be tested – which would require repeating all your phylogeny-informed analyses, ideally twice to account for different phylogenetic hypotheses – or the conclusions about the first crown-group salamander should be strongly deemphasized in the text.

The reasons we refuse to add sirenids into our dataset are three folds. First, living sirenids (Siren + Pseudobranchus) have many autapomorphies (e.g., loss of pelvic girdle + hind limb, separate scapular and coronoid) at the level of Caudata and numerous morphological specializations (e.g., extremely narrow snout, no teeth in upper jaw and dentary, a toothed coronoid) including features specifically related to our manuscript, such as presence of a patch of teeth on vomer and palatine formed by multiple rows of tiny teeth (Reilly and Altig, 1996; and see many datasets on the MorphoSource platform). However, the only known fossil sirenid Habrosaurus found from the latest Cretaceous (late Maastrichtian [72.1-66 Ma]) to Paleocene has a more general configuration in the skull (e.g., elongate maxilla, rudimentary upper jaw, teeth present in upper jaw and dentary; single tooth row on each vomer; but a larger palatine with even denser teeth than living sirenids) when compared to living sirenids (Gardner, 2003: Figure 3I-L; Figure 9), indicating the specialization of sirenids is not formed from the very beginning of their evolution, just like the evolution of morphological specializations of caecilian for their fossorial lifestyle as you mentioned elsewhere. Unfortunately, specimens attributed to Habrosaurus are too poorly-preserved to allow a full restoration of the skull, and the palate remains incompletely known (parasphenoid is unknown Gardner, 2003), leaving it impossible to be added into our dataset. If Sirenidae does share a sister group relationship with Salamandroidea (geologically oldest representative Beiyanerpeton dates back to ~160 Ma), there would be a ~90 Ma fossil gap for Sirenidae that would greatly impact our understandings of their early evolution and the configurations of the palate.

Second, sirenids are probably the only herbivory salamanders (remaining salamanders are carnivory) and have recently been shown as having a unique way of intraoral food processing, which is probably different from all other lissamphibians (Schwartz et al., 2020, 2021): instead of swallowing food unreduced, sirenids have complex three-dimensional chewing behavior to extract energy from plant matters. In our opinion, sirenids might have biomechanical patterns different from all other salamanders during feeding to coordinate with the chewing behavior, and thus their specialized configurations in the palate (such as the enlarged palatine and the many palatine teeth [Gardner, 2003: Figure 3J]. Note that palatine is absent in almost all other salamander clades at the adult stage, except proteids) may receive different selection pressure on the palate of non-sirenid salamanders. The special feeding mode of sirenids and the absence of palatine and palatine teeth in most other salamander clades show that it would be inappropriate to include sirenids into our dataset.

Third, both the extreme specialization in morphology in sirenids and the rampant homoplasies shared with other neotenic taxa (see below) led to the fact that consensus about the phylogenetic position of sirenids has not been reached for over 130 years (Cope, 1889; Larson and Dimmick, 1993; Zhang and Wake, 2009). To date, sirenids are found either as the sister group taxon to Salamandroidea or nested within the latter at different positions.

We also do not agree with the statement “all known sirenids (Cretaceous to extant) are only partially metamorphosed (much like Andrias)”. Sirenids have long been recognized as obligate neotenic species as amphiumids and proteids and share many homoplasies stemming from their aquatic adaptations (e.g., elongate trunk, long and bushy external gills; e.g., Deban and Wake, 2000; Wake and Deban, 2000; Wake, 2009; Bonett and Blair, 2017) and have way more neotenic features than the partially metamorphosed cryptobranchids (Andrias + Cryptobranchus). Cryptobranchids are heavily metamorphosed and retain only few neotenic features, for example, external gills are lost in both Andrias and Cryptobranchus; Cryptobranchus retains gill slits whereas Andrias has the gill slits closed.

Clearly, with so many uncertainties and outstanding questions centering around sirenids, inclusion of sirenids into our dataset will only bring harm, heavily reduce the credibility of many results of our study and will for sure bring unreliable hypotheses including reconstructions of the palate shape.

For Salamandroidea, their fossil records are scarce in the Mesozoic and in our dataset we included the most primitive salamandroid Beiyanerpeton with the hope to add appropriate and relevant data for analyses, such as shape reconstructions of the palate for Urodela.

Some citations above are included in our manuscript and we listed those not included below:

Cope, E.D. 1889. The Batrachia of North America. Bulletin of the United States National Museum 34:1–515.

Gardner, J.D. 2003. Revision of Habrosaurus Gilmore (Caudata; Sirenidae) and relationships among sirenid salamanders. Palaeontology 46:1089–1122.

Larson, A., and W.W. Dimmick. 1993. Phylogenetic relationships of the salamander families: an analysis of congruence among morphological and molecular characters. Herpetological Monographs 7:77–93.

Reilly, S.M., and R. Altig. 1984. Cranial osteology in Siren intermedia (Caudata: Sirenidae): paedomorphic, metamorphic and novel patterns of heterochrony. Copeia 1996:29–41.

Schwartz, D., N. Konow, Y.T. Roba, and E. Heiss. 2020. A salamander that chews using complex, three-dimensional mandible movements. Journal of Experimental Biology 223:jeb220749.

Schwartz, D., M.T. Fedler, P. Lukas, A. Kupfer. 2021. Form and function of the feeding apparatus of sirenid salamanders (Caudata: Sirenidae): three-dimensional chewing and herbivory? Zoologischer Anzeiger 295:99–116.

Zhang, P., and D.B. Wake. 2009. Higher-level salamander relationships and divergence dates inferred from complete mitochondrial genome. Molecular Phylogenetics and Evolution 53:492–508.

Following earlier analyses by the first and the last author, the phylogenetic positions of all mentioned Mesozoic salamanders are simply stated as facts. I'm surprised the work of Rong et al. (2020) is nowhere cited. It showed that the existing datasets for phylogenetic analysis of early salamanders are riddled with too many inaccuracies and redundant characters to be reliable. While Rong et al. (2020) did not undertake the necessary complete review of these datasets, which means their conclusions are not wholly reliable either, they did show that modest improvements result in cladograms that show Chunerpeton (redescribed in that paper), Beiyanerpeton, Qinglongtriton and possibly all other Mesozoic Chinese salamanders outside the salamander crown group. The reconstruction not only of the first crown-group salamander, but also of the first crown-group cryptobranchoid is affected.

We believe the results of any cladistic analyses are hypotheses at best not facts, and we support our hypotheses with evidences at hand like other researchers support their own. And we clearly cited the source references for cladograms we used in this study.

Thank you for bringing up the work of Rong et al. (published online in 2020; printed in 2021). Throughout their article, we did not find any words supporting the above statement “existing datasets for phylogenetic analysis of early salamanders are riddled with too many inaccuracies and redundant characters to be reliable”, and the only relevant contents we found are two sentences below:

1. “For our first analysis, we modified fourteen codings (Appendix A, Section 3) for Chunerpeton in the matrix of Jia and Gao (2019), based on the new fossils we examined” (Rong et al., 2021: p.719)

2. “For our second analysis, we re-coded 23 characters for Jeholotriton and 33 characters for Iridotriton (Appendix A, Section 3), based on details in relevant publications (Evans et al., 2005; Wang and Rose, 2005; Carroll and Zheng, 2012), whereas both taxa were excluded in preceding study (Jia and Gao, 2019)” (Rong et al., 2021: p.720)

We are happy to talk about our interpretations of the cladistic analyses done by Rong et al. (2021) and show how their results do not impact our work: Rong et al. (2021) conducted a total of five different phylogenetic analyses, and their Figure 5A and Figure 5B show the result of their first and second analyses, respectively. By taking a very close look at Figure 5, we see that not only were Beiyanerpeton and Qinglongtriton not recovered as basal salamandroids, but neither Hynobiidae nor Cryptobranchoidea were recovered as monophyletic groups. These results from Figure 5 of their work certainly invite skepticism because not only have Beiyanerpeton and Qinglongtriton been supported as basal salamandroids by many diagnostic characters of Salamandroidea as recovered from our original cladistic analyses (Gao and Shubin, 2012; Jia and Gao, 2016; see other responses below), but also the monophyly of both the Hynobiidae and Cryptobranchoidea have been well established by studies using molecular data alone, morphological data alone, and studies combining both molecular and morphological data (summarized in our work on Nuominerpeton; https://peerj.com/articles/2499/).

Rong et al. (2021:p.720; or at the bottom left on the page with Figure 7) also expressed their lack of confidence in the results of the first two cladistic analyses by saying: “Because the family-level relations of living salamanders recovered in our first two analyses are inconsistent with molecular results (cf. Figure 5A, B versus Figure 6B), we performed a third analysis in which relationships of recent families were constrained by the molecular tree (Pyron and Wiens, 2011, Figure 6B)”.

As shown in Figure 6A of Rong et al., 2021, their third analysis not only recovered Qinglongtriton and Beiyanerpeton as basal members of Salamandroidea, Regalerpeton as stem hynobiid, but also successfully found the monophyly of Cryptobranchoidea, although hynobiids still failed to form a clade as admitted by Rong et al., 2021 (see P.720, or the paragraph on the right side of Figure 7). Chunerpeton was found as forming a polytomy with two other clades of crown urodeles. These results are more consistent with previous studies including Rong, 2018. Their fourth (as shown in their Figure 6C) and fifth (as shown in their Figure 7) analyses were conducted following the same strategy as in their third analysis, but most fossil salamander taxa including Beiyanerpeton and Qinglongtriton were excluded, and therefore these results are not helpful in understanding the real phylogenetic position of many fossil taxa including Chunerpeton.

Rong et al. (2020) further pointed out the nomenclatural fact that "herpeton" is grammatically neuter and that therefore the International Code of Zoological Nomenclature automatically corrects a number of species names from "-is" (masculine or feminine) to "-e" (neuter). The authors and dates of the names are not affected by this.

That is correct. We cited Rong et al., (2021) for this purpose in our Materials and methods section as below: “To keep the gender of species names consistent with that of genus names as per ICZN codes, we replaced the feminine/masculine species ending (“-is”) by corresponding neuter forms (“-e”) for genus names (e.g., Nuominerpeton) end with the neuter noun “herpeton” or “ἑρπετόν” in Greek as suggested in Rong et al. (2021).”

Species named after localities usually ends with “ensis” and does not create problems when the ending of genus names is either feminine or masculine, because “ensis” has the same form for both feminine and masculine. Considering that the Greek word “herpeton”, “ἑρπετόν”, is neuter in grammar and when it is appended to the genus name the corresponding species name should be neuter as well. The neuter form of the ending “ensis” is “ense” and now we have revised the species name ending with “ensis” by “ense” for genus names ending with “erpeton” throughout the manuscript and the supplementary file 1, including: Beiyanerpeton, Chunerpeton, Nuominerpeton, Pangerpeton, and Regalerpeton.

You follow common usage among paleontologists since the early 1990s in calling the crown group of salamanders Urodela and the total group Caudata. Apparently without talking to anyone, Wake (2020) has defined the name Caudata as applying to the crown group of salamanders in a way that is valid under the International Code of Phylogenetic Nomenclature (Cantino and de Queiroz, 2020). Wake (2020) explicitly left the name Urodela undefined. It might be best if you mention this situation in a few words in the manuscript. In the longer run, beyond this manuscript, it would probably be best to ask the Committee on Phylogenetic Nomenclature for an emendation of the definition of Caudata. I'm a member of the Committee and would happily coauthor a paper for this purpose with you and other experts on salamanders.

As you said, most researchers are now using Caudata to represent the total group salamanders and Urodela the crown group salamanders, and only few works used the term the other way around (e.g., Schoch, 2020). All of our four text figures have explicitly labeled total group salamanders as Caudata and crown group as Urodela, and we feel no need to stress/advertise somewhere in our text the “not popular” way of usage proposed by Wake (2020). ICPN is gaining momentum in the Era of cladistics and we appreciate your invitation to coauthor a paper to address the usage of Caudata/Urodela. Let’s stay in touch and chat over ideas on how to proceed with this project.

Lines 30, 40: I don't think Chinlestegophis and Rileymillerus should be accepted as undoubted caecilians. But as it happens, an undoubted Late Triassic caecilian was recently announced in a published conference abstract by Kligman et al. (2021), so the oldest known caecilians are Late Triassic in age either way.

Thank you for letting us known the new discovery of another Late Triassic caecilian. We kept the citation “Pardo et al., 2017a” (which is the original study proposing the stem caecilian affinities for Chinlestegophis and Rileymillerus) in the first sentence of Introduction Section, and at the same place we added “Kligman et al., 2021” and updated the reference list to strengthen our statement of the oldest known caecilian dates back to the Triassic.

35: These two papers are neither the most recent nor in any other sense the most important ones on this subject. I recommend citing Pardo et al. (2017b) and Daza et al. (2020: Figure 4D, E, S13, S14) instead.

We kept the original two citations (Fröbisch and Schoch, 2009; Maddin and Anderson, 2012) because both papers are relevant in paleoecological interpretations of dissorophoid temnospondyls, which are really our focus in this sentence as well as this manuscript. We also added “Pardo et al., 2017b” here as suggested, however we did not add “Daza et al., 2020” because there are so many uncertainties in their numerous phylogenetic hypotheses, for instance salamander species Chelotriton, karaurids and other urodeles were often recovered as not forming a monophyletic clade (figures 4E, 4F, S14, S15). Most importantly, Daza et al., 2020 contains no information on the paleoecological interpretations of purported ancestor groups of lissamphibians.

36: "2017b" is an error for "2017a".

Our mistake. We replaced “2017b” with “2017a”.

37: The review paper by myself and Laurin (2013) is not up to date, being written long before completion of the large phylogenetic analysis by myself and Laurin (2019), let alone its update by Daza et al. (2020: Figure 4F, S15), not to mention the analysis of ontogeny by Laurin et al. (2022). Importantly, it is clear that Lissamphibia is not derived from aquatic lepospondyls.

As you can tell in this sentence what we are really emphasizing is the paleoecological interpretations of the potential ancestral groups of lissamphibians, and you and Laurin’s work published in 2013 contains sufficient information (both information on hypotheses on Lissamphibia origin and paleoecological preferences of lepospondyls) we needed. But here we are happy to keep our work more up-to-date by citing your other works (Marjanović and Laurin, 2019; Laurin, Lapauze and Marjanović, 2022).

40-42: This is plainly not true. Chinlestegophis obviously lived in burrows, but apart from its elongate body shape it shows no adaptations to burrowing. Even the Early Jurassic Eocaecilia and, as far as its fragmentary remains allow us to tell, the Early Cretaceous Rubricacaecilia lack some of the crown group's adaptations to burrowing, e.g. they retain limbs, larger orbits and more sutures in the skull.

Thanks for letting us know your different opinion on Chinlestegophis’s morphological adaptations for burrowing. However, besides body elongation several other morphological specializations to support the burrowing mode of life for Chinlestegophis were listed in the original study (cited in our manuscript as Pardo et al., 2017a: p. E5393): “the consolidation of the skull, reduction of the orbits, and anteriorization of the jaw articulation suggests that Triassic stem group caecilians were increasingly specialized for life and feeding in confined spaces”. To remove any confusions, we revised this sentence as “…caecilian Chinlestegophis from the Triassic have displayed several morphological specializations as their living relatives…”

49: Of any two sister groups, each is more primitive than the other in some respects but not in others. It makes little sense to call Cryptobranchoidea "the most primitive clade of the crown group salamanders". I suggest "the sister group to all other crown salamanders".

We agree with your first sentence in the comment but our original sentence at Line 49 has nothing to do with the sister group of Cryptobranchoidea, instead the sister-group taxa within Cryptobranchoidea is what we were focusing on.

The crown group salamanders, Urodela, are traditionally classified into three suborders: Cryptobranchoidea, Sirenoidea and Salamandroidea. Our comparison was based on these three clades. Cryptobranchoidea is usually found as the sister-group taxon to Sirenoidea + Salamandroidea, but Sirenoidea is sometimes found by other studies as nested within Salamandroidea in different places. Moreover, Cryptobranchoidea has many more plesiomorphic features (e.g., two centralia in the mesopodium) than Sirenoidea and Salamandroidea. In this regard and considering the uncertain phylogenetic positions of Sirenoidea, we kept the sentence the way it is.

57, 625-626: For the life history of Aviturus, see Skutschas et al. (2018).

Thank you for reminding us of this paper. The main conclusion of Skutschas et al. (2018)’s work on the zygapophyseal skeletochronology of Aviturus is that this fossil taxon shares with modern cryptobranchids by having a similar growth rate. This conclusion took us a step closer to the lifestyle of Aviturus, however neither the ecological preference nor life history strategy (neoteny/metamorphosis) of Aviturus was explicitly stated. We added this citation at Line 57 and Lines 625-626 after “Vasilyan and Böhme, 2012” and updated our reference list.

58-59: It is not excluded that Regalerpeton is a stem-group salamander (Rong et al. 2020: Figure 5).

see our above responses.

71, 73: Two more good opportunities to cite Rong et al. (2020).

89, 173-179, 446-450, 615: but see Rong et al. (2020) for reasons for skepticism.

For the second comment please see our responses above. Below are our responses to the first comment.

The reason we refused to cite Rong et al. (2021) at the suggested places is that we can clearly see that the real problem of Rong et al., 2021’s redescription on the so called “Chunerpeton tianyiense” was mainly based on “31 referred fossil skeletons” (see P.709 in the Materials and methods Section) that have a “snout-pelvic length ranging from 20 mm (IVPP V 13241A&B) to 115 mm (IVPP V 15422)” (see P. 710 in the first paragraph of section “4.1 General features”). As you can tell from their Material and methods section, most specimens are incomplete (see Rong et al., 2021: p.710). Indeed, their anatomical interpretations and line drawings are mainly based on a juvenile form (IVPP V13343; see Rong et al., 2021: figures 1 and 2) with a snout-pelvic length of about 90 mm and a total length less than 110 mm, and several other even smaller specimens (IVPP V 14226A as in Figure 3A; IVPP V 15422 as in Figure 3B) if you compare their skull length as shown in Figure 2 and Figure 3A and 3B. The specimen IVPP V13343 and other specimens display juvenile features, including but not limited to the presence of a frontoparietal fontanelle, weakly ossified and loose articulation patterns seen in the phalanges (Figure 3G, 3H), and the weakly ossified epiphysis in the hindlimb (Figure 3I, 3J). Moreover, an ossified orbitosphenoid was admitted (Rong et al., 2021:p.715, at the bottom left) to “form the bony lateral wall of braincase in most mature salamanders except proteids (Rose, 2003)” and such an ossified orbitosphenoid is “not observed…in any of our specimens”. By contrast, the holotype specimen (total length ~ 180 mm) of Chunerpeton tianyiense lacks the frontoparietal fontanelle, has an ossified orbitosphenoid (termed as “hypohyal” in type description) and has the humerus almost in contact with the radius/ulna. It is also important to note an ossified orbitosphenoid is absent in the 46 reported specimens of basal salamandroid Qinglongtriton including many fully-grown adults, reinforcing that the ossification of orbitosphenoid in early salamanders is taxonomically informative (unlike the consistent presence in modern salamanders as stated by Rose, 2003), not to mention the so many other anatomical differences in Chunerpeton, Beiyanerpeton and Qinglongtriton (such as presence/absence of spinal nerve foramina in the vertebrae, which are important feature to differentiate Cryptobranchoidea, Salamandroidea and Sirenoidea; see Jia and Gao, 2016).

Salamander morphologies are greatly impacted by development, and only those morphological features in adults are trustworthy for taxonomic and phylogenetic interpretations. As pointed out in our previous review work (Gao et al., 2013), neotenic taxa tend to be large in body size. It is unfortunate to see Rong et al., 2021 draw their conclusions based on juvenile specimens, while reading that “Chunerpeton is a large fossil salamander, with some individuals reaching body lengths of up to 50 cm (~18 cm in the holotype)” in another paper (Sullivan et al., 2014: p.250) co-authored by the senior author in Rong et al., 2021. Based on so many anatomical differences from the holotype of Chunerpeton, it is quite likely that the batch of specimens studied by Rong et al., 2021 represent a new species that was misclassified as Chunerpeton tianyiense.

Anyway, we are reluctant to distract our present study by including so many uncertainties introduced by Rong et al., 2021’s work to test their conclusions. But we feel obligated to make clarifications and will hopefully address these problems with our own specimens of Chunerpeton tianyiense in the near future, but this is not within the scope of our current study.

References not listed in the manuscript:

Sullivan, C., Y. Wang, D.W.E. Hone, Y. Wang, X. Xu, and F. Zhang. 2014. The vertebrates of the Jurassic Daohugou Biota of northeastern China. Journal of Vertebrate Paleontology 34:243–280.

110, 198, 268, 271, 276, 277, 279, 280, 282, 284, 286, 289, 290, 334, 337, 428: Insert "last" before "common"!

Good point, done.

194: Replace "albert" by "albeit" (or "although" or just "though").

Our typo. Thank you!

202, 602, 635: Uppercase for Procrustes (the name of a mythological person).

“p” is now capitalized for “Procrustes” throughout the text.

293-294: All adult anurans and caecilians are metamorphosed, but some of both are fully aquatic (e.g. Pipidae, Typhlonectidae). I would therefore rearrange the sentence to: "Among modern amphibians, the adults of anurans and caecilians are metamorphosed, and most of them are terrestrial."

Thanks for the suggestion. We refused to add “the adults of anurans and caecilians are metamorphosed” considering that the life history strategy of many caecilians is direct development, which is different from metamorphosis because there is no two-phased development after hatching for direct developers. We kept the original sentence and added “mostly” between “their postmetamorphosed adults are” and “terrestrial” to cover the exceptions of the few aquatic taxa.

299: …or whatever will remain of Lepospondyli.

308: I would write "metamorphic taxa" or "metamorphosed individuals".

We chose the latter suggestion.

339-340: Aviturus, which dates from the very end of the Paleocene, is not the earliest known pancryptobranchan. The earliest entirely undoubted one is "Cryptobranchus" saskatchewanensis, which is a few million years older (late middle Paleocene). There is evidence that the much older (mid-Cretaceous) Eoscapherpeton is a stem-pancryptobranchan; see Marjanović (2021: supplementary material pp. 13-16) for references and a brief review.

Thanks for the correction and reminding of your summary on Eoscapherpeton and other fossil cryptobranchids. We now have the sentence “The earliest known fossil pancryptobranchan Aviturus” replaced as “The Paleocene pancryptobranchan Aviturus”.

480: The plural of pars palatina is partes palatinae – as in most languages of Europe (but unlike English), number and gender (and case) are marked on adjectives as well as nouns in Latin.

Thank you so much for correcting our usage on the plural form of “pars palatina” from “partes palatina” to “partes palatinae” and please find more of our responses to this issue in our replies to Reviewer #1.

484: Replace "pas" by "pars".

Thanks for pointing out the typo. We replaced “pas” by “pars”.

791: Replace "batrachian" by "batrachians".

done.

857-859: Unlike English, German does not have separate capitalization rules for headlines – but it always gives a capital Letter to every Noun, while almost nothing else is ever capitalized, not even Adjectives derived from proper Names. Therefore, please correct the Title to: "Generelle Morphologie der Organismen: Allgemeine Grundzüge der organischen Formen-Wissenschaft, mechanisch begründet durch die von Charles Darwin reformirte Descendenz-Theorie".

Revised. Good to know.

Finally, the supplementary file claims to be a PDF file ("This PDF file contains supplementary file 1A to 1K."), but it is a DOCX file. In the interest of wider accessibility, I recommend you convert it to PDF before resubmitting it.

Done.

I hope these comments are helpful. I apologize again for the delay, and I'm looking forward to the next version of your manuscript!

References not cited in the manuscript:

Cantino PD, de Queiroz K. 2020. International Code of Phylogenetic Nomenclature (PhyloCode). Version 6. CRC/Taylor & Francis/Informa, Boca Raton/London/New York. ISBN: 978-1-138-33282-9 (paperback), 978-1-138-33286-7 (hardback), 9780429446320 (e-book). DOI: https://doi.org/10.1201/9780429446320 Openly accessible at http://phylonames.org/code/

Daza JD, Stanley EL, Bolet A, Bauer AM, Arias JS, Čerňanský A, Bevitt JJ, Wagner P, Evans SE. 2020. Enigmatic amphibians in mid-Cretaceous amber were chameleon-like ballistic feeders. Science 370:687-691. DOI: https://doi.org/10.1126/science.abb6005

Kligman B, Stocker M, March A, Nesbitt S, Parker W. 2021. New Late Triassic stem-caecilian from southwestern North America strengthens evidence for lissamphibian monophyly, and illuminates the anatomical, functional and geographic origins of living caecilians [abstract]. Society of Vertebrate Paleontology (ed.): Virtual meeting conference program, 81st annual meeting, p. 160. The entire abstract volume can be downloaded here: https://vertpaleo.org/svp_2021_virtualbook_final/

Laurin M, Lapauze O, Marjanović D. 2022. What do ossification sequences tell us about the origin of extant amphibians? Peer Community Journal 2:e12. DOI: https://doi.org/10.24072/pcjournal.89

Marjanović D. 2021. The making of calibration sausage exemplified by recalibrating the transcriptomic timetree of jawed vertebrates. Frontiers in Genetics 12:521693. DOI: 10.3389/fgene.2021.521693

Marjanović D, Laurin M. 2019. Phylogeny of Paleozoic limbed vertebrates reassessed through revision and expansion of the largest published relevant data matrix. PeerJ 6:e5565. DOI: https://doi.org/10.7717/peerj.5565

Rong Y-F, Vasilyan D, Dong L-P, Wang Y. 2020 (printed 2021). Revision of Chunerpeton tianyiense (Lissamphibia, Caudata): Is it a cryptobranchid salamander? Palaeoworld 30:708-723. DOI: https://doi.org/10.1016/j.palwor.2020.12.001

Skutschas PP, Kolchanov VV, Bulanov VV, Sennikov AG, Boitsova EA, Gulbev VK, Syromyatnikova EV. 2018 (printed 2020). Reconstruction of the life history traits in the giant salamander Aviturus exsecratus (Caudata, Cryptobranchidae) from the Paleocene of Mongolia using zygapophyseal skeletochronology. Historical Biology 32:645-648. DOI: https://doi.org/10.1080/08912963.2018.1523157

Wake DB. 2020. Caudata J. A. Scopoli 1777 [D. Wake], converted clade name. [Brackets in the original.] de Queiroz K, Cantino PD, Bauthier JA (eds): Phylonyms. A Companion to the PhyloCode (CRC/Taylor & Francis/Informa, Boca Raton/London/New York), pp. 785-787. ISBN: 978-1-138-33293-5 (hardback), 9780429446276 (e-book). DOI of the entire book: https://doi.org/10.1201/9780429446276

[Editors' note: further revisions were suggested prior to acceptance, as described below.]

The manuscript has been improved but there are some remaining issues that need to be addressed, as outlined below:

Reviewer #3:

The issues with image quality in the merged PDF have disappeared. Please correct the species names in figures 1, 3, 19, 23-26, 30-32, 36 and 38 if I've counted correctly, and replace "semiaqaic" with "semiaquatic" in Figure 3.

Correct species names are now labeled in Figures 1, 3; and the many supplementary figures: Figure 1—figure supplements 15, 19-22, 24, 26; Figure 2—figure supplements 1, 5; and Figure 3—figure supplement 2. Our typo was also corrected in Figure 3.

Sirenidae and the first crown-group salamander.

Your anatomical reasons for the omission of sirenids are convincing, and I had indeed confused sirenids and amphiumids in my statement about partial metamorphosis – thank you for pointing this out! I am, however, quite surprised that you present the phylogenetic position of Sirenidae as a mystery. Sirenidae and Salamandroidea have been found as sister groups in every large-scale study of molecular data, no matter which data or which method exactly: 7,189 transcripts of nuclear genes and Bayesian inference (Irisarri et al. 2017; 99% posterior probability), 5 mitochondrial and 10 nuclear genes and maximum likelihood (Vijayakumar et al. 2019: supplementary file Amphibia_New_India_SHL_Dryad.tre; 52% bootstrap support – note this is the latest and largest version of R. A. Pyron's series of matrices, and all versions, including Jetz and Pyron [2018] which you cite, found the same topology), or 120 nuclear protein-coding genes, separately and together, and maximum likelihood (Himes et al. 2020: 100% bootstrap and ASTRAL posterior values). Given such extraordinary agreement, the fact that the morphological evidence is less clear can be blamed on the morphological evidence, or on our lack of knowledge of it (I greatly appreciate your recent work in describing the osteology of extant salamanders!), but it would be very difficult to try to argue the molecular data away. Therefore, I think you should mention in the text that you cannot include Sirenidae in your dataset for the reasons you presented in your response and that this is a potential weak point in inferences about the last common ancestor of all crown-group salamanders.

We thank you for your summary on the recent molecular cladistic analyses and their conclusions on the phylogenetic position of sirenids. Based on our last responses, we now have added a few sentences in the first section of Materials and methods titled “Experimental design, specimens, palate” to explain why sirenids were not included in our dataset.

You point out that the analyses of Rong et al. (2020, printed 2021) failed to find Hynobiidae or Cryptobranchoidea. Unfortunately, this is not incompatible with their matrix being an improvement over its sources. It is not uncommon (e.g. Marjanović and Laurin, 2019, and references therein) for improvements to matrices for phylogenetic analysis to decrease resolution, and it is not uncommon either for matrices to support the right things for the wrong reasons. Matrices that support clades for the wrong reasons are unreliable in the sense that they cannot be trusted to place added taxa accurately. As I stated, the version by Rong et al. (2020) cannot be relied upon as strong evidence for the phylogenetic positions of Chunerpeton, Beiyanerpeton or any other taxon – but that does not make the earlier versions of that matrix any better. They contain, after all, such phenomena as redundant and even duplicate characters: for example, the haploid and the diploid number of chromosomes are both included as separate characters in the previous versions. Duplicating a character inevitably distorts, if not the topology, then at the very least the support values for that topology (references in Marjanović and Laurin, 2019: 15-16). Rong et al. (2020) made a certain effort to reduce this problem.

They also ordered certain characters, unlike their sources. Ordering is widely believed to be a philosophical question, but there is strong evidence from simulations as well as from empirical studies that potentially clinal or meristic characters must be ordered to avoid inaccurate results as well as both false negatives and false positives in resolution (references and brief discussion in Marjanović and Laurin, 2019: 16).

The matrix of Rong et al., 2021 is an improvement of the source dataset (Jia and Gao, 2019 which in turn is based largely on Gao and Shubin, 2012) in terms of recoding certain characters for Jeholotriton, Iridotriton and Chunerpeton based on their own reinterpretations and inclusion of molecular trees as backbones. We checked this paper carefully and, unfortunately, we didn’t find they “ordered certain characters” because they stated it clearly in the first paragraph in Section 6.3---“We designated the stem salamander Karaurus as outgroup and set all characters as unordered and equally weighted” (page 719). But experimenting analytic strategies like ordering certain characters as you mentioned above should definitely be encouraged, especially when developmental trajectories of certain characters are well understood. Therefore, more studies on relevant extant taxa and growth series as we are working on will be potentially helpful to increase sampling in taxa and characters for cladistic analyses.

Our original arguments in previous responses are that their results from the first two cladistic analyses (where monophyletic status for Hynobiidae and Cryptobranchoidea was not recovered) of Rong et al. (2021) were even doubted by the authors themselves, and their several inferences including the one you mentioned in your previous comments (Beiyanerpeton, Qinglongtriton, possibly all other Mesozoic Chinese salamanders outside the salamander crown group) are paradoxically based on their first two cladistic analyses. However, these inferences can not be drawn from their third cladistic analysis, which includes a molecular tree as backbone and is also favored by the authors.

Phylogenetics with morphological data

Parsimony analysis of morphological data should not be treated as a black box.

Daza et al. (2020) did all their phylogenetic work on previously published matrices; they added or updated the scores of Albanerpetidae, but did not make any other changes to those matrices. Therefore, practically all the problems with their results are the fault of those matrices. The clearly wrong placement of Chelotriton in their Figure 4F and S15 stem from the fact that I had added Chelotriton to the source matrix in question, that of Marjanović and Laurin (2019), which is simply not equipped to handle crown-group salamanders. (That matrix is an attempt to improve the one by Ruta and Coates, 2007. In order to limit the amount of time and effort, we did not add any characters to that matrix. The matrix of Ruta and Coates, 2007, contained only two salamanders – Karaurus and Valdotriton – and therefore lacked any characters specific to salamander phylogeny.) I did this in order to test a point on ontogeny and phylogeny: would the extreme metamorphosis of Chelotriton, which makes it look more like a temnospondyl than lissamphibians usually do, pull some or all lissamphibians into Temnospondyli? That did not happen, but Chelotriton was pulled out of the salamander clade. The latter result is obviously wrong, and obviously due to the lack of salamander-specific characters in the matrix; there is no reason to think that it indicates a more general problem. The former result, on the other hand, is actually strengthened that way.

In another analysis, Daza et al. (2020: Figure 4E, S14) found Karauridae outside Batrachia. I haven't looked into its precise causes, but it is simply shared with the source matrix of that analysis, the matrix of Pardo et al. (2017a).

Both of these analyses are, however, relevant to the ecology of the first lissamphibian by finding Albanerpetidae as the sister group of Lissamphibia, Albanerpetidae being terrestrial and fully metamorphic or of course direct-developing (early juveniles of any sort are not known). This is a data point for phylogenetic bracketing.

Thank you for sharing with us the backstories of the cladistic analyses done by Daza et al., 2020.

In the context of the specimens referred to Chunerpeton by Rong et al. (2020), you write: "only those morphological features in adults are trustworthy for taxonomic and phylogenetic interpretations." In phylogenetics, however, morphological features are not automatically comparable just because they are found in sexually mature adults, as you seem to have assumed in your published phylogenetic work: for much of the skeleton, for instance, neotenic adults are much more easily comparable to larvae than to adults of metamorphic taxa, so that the presence or absence of many character states of metamorphic adults must be scored as unknown/inapplicable in neotenic taxa. This has a strong effect on the topologies found by phylogenetic analyses (Wiens et al., 2005; Marjanović and Laurin, 2019: 21-22, and references therein).

What we argued are that development in salamanders have enormous impacts on their morphological features, and states of characters in larval/juvenile/subadult specimens will eventually be replaced or at least modified by states of characters observed in sexually mature specimens, and therefore the final, stable state of a character is only found in sexually-mature specimens regardless of whether the species is neotenic, metamorphosed or direct developing. Even in neotenic salamanders, in which morphological changes during development are fewer than that in metamorphosed or direct developing taxa, certain characters such as proportion, shape, ossification extent will change developmentally and we need to maximally alleviate effects on phylogeny/taxonomy from ontogeny by scoring characters based on adult specimens.

Comparing character states displayed by adult specimens from species in different lifestyles (e.g., neoteny, metamorphosis), as what you emphasized above, is indeed a different story. We agree that certain morphological features are retained from larvae to adults in neotenic taxa and otherwise are resorbed during metamorphosis and thus cause incomparable phenomena for neotenic and metamorphic/direct developing taxa. But these phenomena should not be deemed as reasons to not score characters from adult specimens but instead should be creatively dealt with analytical strategies such as coding neotenic characters as “?” or character ordering or weighting.

In this case, the so-called “referred specimens” to Chunerpeton tianyiense are all juveniles based on several evidences we listed in previous responses (e.g., incomplete ossification of epiphyses of long bones). Without adult specimens, the taxonomic affiliation of these juveniles is truly unknown. One possibility as we mentioned in our previous response is that these juveniles may represent a new species of Chunerpeton because they have displayed differences with the holotype specimen of C. tianyiense (e.g., orbitosphenoid present & absent). Another possibility is that theoretically theses juvenile specimens may simply represent not-yet-metamorphosed-individuals of an unknown, metamorphic species, because neotenic taxa in fossil record can only be distinguished by the presence of larval features in adult specimens.

Nomenclature and taxonomy

I greatly appreciate your comments on the validity of the referral of the new specimens by Rong et al. (2020) to Chunerpeton, and I'm looking forward to your further publications on this matter!

Likewise, I appreciate your comments on the definitions of the names Caudata and Urodela and will try to send you a draft manuscript as soon as possible. For the purposes of the present manuscript, I withdraw all my objections.

Thank you for your invitation and we look forward to working with you on this interesting manuscript! We have to say, as you may have already noticed, that Evans and Milner (1996) have provided a brief “Taxonomic note” in their paper (p. 629) describing Valdotriton explaining the usage of “Caudata” and “Urodela” before 1988 and after 1988. We will be prepared for your manuscript and try to dig up the old literatures for the usage of these two terms.

Evans SS, Milner AR. 1996. A metamorphosed salamander from the Early Cretaceous of Las Hoyas, Spain. Philosophical Transactions: Biological Sciences 351:627-646. URL: http://www.jstor.org/stable/56320

Adaptations of Chinlestegophis

Thank you for reminding me of the features listed as adaptations to burrowing by Pardo et al. (2017a). I agree that the fairly small orbits may count as such, though they could also hint at life at the bottom of muddy bodies of water – a strictly aquatic lifestyle, apart from sheltering in burrows with 100% humidity, is assured by the lateral-line grooves. The position of the jaw articulation, however, is shared with all other brachiopods, where it is an adaptation to a particular style of suction feeding; it is in fact more extreme in Batrachosuchus, depicted in Pardo et al. (2017a: Figure 3). By "consolidation of the skull", Pardo et al. (2017a: supp. inf. part F) mean the frankly irrelevant fusion of lacrimal and maxilla – besides, it is not in fact clear whether a lacrimal is even present -, the supposed fusion of the pterygoid and the quadrate for which there is very little evidence presented in the paper, and fusion of the exoccipitals and the basioccipital which is universal in dissorophoid temnospondyls, none of which were burrowing or had any sort of digging lifestyle – and note that it is not clear if a basioccipital was present in the first place; it remained cartilaginous and very small in other brachiopods. In a borrower, one would expect, as in extant caecilians, a well-ossified braincase that could function as a strut to enable the skull to resist rostrocaudal compression, dorsoventral bending, or twisting; yet, most of the braincase is not ossified at all in Chinlestegophis (Pardo et al. 2017: supp. inf. part B). This is particularly striking in comparison to the burrowing "lepospondyls" described by Pardo and various coauthors in the two years prior.

Interestingly, the stem-caecilian presented by Kligman et al. (2021) seems not to be adapted to burrowing at all, but it is clearly much closer to the crown group than Chinlestegophis.

Thank you for sharing with us your thoughts on the burrowing adaptation in early caecilians.

Details

Lines 38-41: I had not quite appreciated that here you cite references both for the lifestyles and the phylogenetic positions of the listed taxa, and only suggested references for the phylogenetic positions of various "lepospondyls". For the terrestrial lifestyle of at least one amphibamid, I recommend Laurin et al. (2004) and references therein. For the various lifestyles of "lepospondyls", I recommend Jansen & Marjanović (2021) and references therein. While some stereospondyls have occasionally been considered semiaquatic for vaguely articulated reasons, almost all were certainly fully aquatic as shown by the lateral-line grooves on their skulls and further supported by their very slow peri- and endochondral ossification; I recommend the brief but clear statement in Schoch & Milner (2014: 123) and references therein. Notably, Chinlestegophis has lateral-line grooves (Pardo et al., 2017a), showing that it was not semiaquatic; "reduced" grooves as identified by Pardo et al. (2017a) mean that most of the lateral-line organ was situated in the skin and did not contact the bone, not that the organ was "reduced" – something that does not occur, because the lateral-line organ dries up and dies from serious exposure to air as inevitably caused by a semiaquatic lifestyle. – While Jansen & Marjanović (2021) is published on a preprint server with the usual disclaimer, it is in fact an accepted manuscript published with permission from the journal (Comptes Rendus Palevol) that accepted it after peer review. The journal is currently changing publishers, a process that started before the pandemic and is still not complete; we've been waiting for the page proofs since March 2021.

Thank you for providing us more literatures on the lifestyles of early tetrapods. We now have added “Laurin et al., 2004”, “Jansen and Marjanović, 2021” and “Schoch and Milner, 2014”. We also replaced “semiaquatic” by “semiaquatic/aquatic” for stereospondylian at Line 40.

80-81: Other than its probable membership in Karauridae, is there evidence on the lifestyle of Marmorerpeton?

As we originally stated here Marmorerpeton was argued to be neotenic by Evans et al., 1988 and many later studies especially those done by Dr. Pavel Skutschas and his colleagues.

85, 106: If you use Caudata as the name for the total group, Triassurus is a stem caudate, not a stem urodele; nothing is a stem urodele, because if something is on the stem from which Urodela comes, it is outside Urodela by definition. At least for the first few decades of this terminology, a clade consisted of a crown group and a stem-group; to be a stem urodele, something has to be a urodele.

We do not agree with the above argument. We tend to believe Caudata (total group) and Urodela (crown group) are two node-based clades. Many taxa including those we included in the Introduction, such as Marmorerpeton, Karaurus, Urupia are stem urodeles because they lack the spinal nerve foramen on atlas that is otherwise present in Urodela. Triassurus is a salamander and it does not belong to the crown group salamander, Urodela, but is located within the total group salamander, Caudata. So we can call it basal caudate or stem urodele to ensure people understanding its rough phylogenetic position. If we designate Triassurus to be stem caudate as suggested here, it then will not a salamander but instead a “salamander-morph”. Unfortunately, in the PNAS paper of Schoch et al. (2020) where Triassurus was restudied based on a second larval specimen, Caudata and Urodela were designated as crown and total group salamanders, respectively, and again unfortunately, Triassurus was called to be a “stem caudate”. Moreover, because of the larval stages of both the holotype and the second specimen, Triassurus remains enigmatic in many aspects (e.g., life history strategy), and states of many characters in fully-grown specimens of Triassurus remain unclear, including the presence/absence of spinal nerve foramina on atlas.

106: Likewise, if you use Urodela as the name for the crown group, "crown urodeles" is redundant; "urodeles" would be enough.

The original sentence is “based …on the palate of all living and …fossil genera of cryptobranchoids, stem and other basal crown urodeles ….”. The reason we kept the word “crown” is we want to make it clear to the readers that we investigated both stem urodeles and basal crown urodeles.

623-624: I would rather write: "As implied by Jia et al. (2021a), we apply the name Hynobiidae to the crown group of Panhynobia".

We replaced “The family Hynobiidae is designated here as crown group Panhynobia” with “Following Jia et al. (2021a), we apply the name Hynobiidae to the crown group of Panhynobia”.

912: "lepospondyl", not "lepospondyls".

The second “s” in “lepospondyls” is now deleted.

928: "Kuro-o", a Japanese name with three syllables; "oo" could be misunderstood as a long vowel – Japanese distinguishes long from short vowels. Sometimes apostrophes are used to disambiguate in transcriptions, sometimes hyphens are used instead.

We replaced “Kuro-O” by “Kuro-o”.

976: "Syromyatnikova".

Done.

977: italics for the genus & species name (present in the original, I've checked)

Done.

Suggestions on style and language

Lines 15-18: "but the small number of reliable ecological indicators established so far hinders investigations into the paleobiology of early salamanders. Here we statistically demonstrate, by using time-calibrated phylogenetic trees and geometric morphometric analysis on 71 specimens in 36 species, that both the shape"…

There are indeed small number of reliable ecological indicators in salamanders, but these characters are from soft tissues such as external gill, caudal fin, digit web in hand and foot that are rarely preserved in fossil record particularly in metamorphosed taxa. No osteological characters that are potentially informative in ecological preferences has been investigated or tested with fossil taxa except the palate studied here. Therefore, we retained the original sentence “but the yet established reliable ecological indicators from bony skeletons hinder investigations into the paleobiology of early salamanders.”

21: I'm not sure what you mean by "strictly". Perhaps "analyzed in detail" would be clearer?

“strictly analyzed” is now replaced with “analyzed in detail”.

25-27: That would mean the disparities, not the salamanders, have achieved the ecological preferences. Would the following be an improvement? "Salamanders began to diversify ecologically before the Middle Jurassic and achieved all their present modes of life in the Early Cretaceous."

We replaced the original sentence into “Salamanders are diversified ecologically before the Middle Jurassic and achieved all their present ecological preferences in the Early Cretaceous”.

https://doi.org/10.7554/eLife.76864.sa2

Article and author information

Author details

  1. Jia Jia

    1. School of Earth and Space Sciences, Peking University, Beijing, China
    2. State Key Laboratory of Palaeobiology and Stratigraphy (Nanjing Institute of Geology and Palaeontology, CAS), Nanjing, China
    3. Department of Comparative Biology and Experimental Medicine, University of Calgary, Calgary, Canada
    Contribution
    Conceptualization, Data curation, Formal analysis, Funding acquisition, Investigation, Methodology, Project administration, Resources, Software, Supervision, Validation, Visualization, Writing - original draft, Writing – review and editing
    For correspondence
    jia.jia@ucalgary.ca
    Competing interests
    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-8243-0156
  2. Guangzhao Li

    Department of Microbiology, Immunology, and Tropical Medicine, The George Washington University, Washington D.C., United States
    Contribution
    Formal analysis, Methodology, Software, Validation
    Competing interests
    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-5007-8338
  3. Ke-Qin Gao

    School of Earth and Space Sciences, Peking University, Beijing, China
    Contribution
    Data curation, Funding acquisition, Resources, Writing – review and editing
    For correspondence
    kqgao@pku.edu.cn
    Competing interests
    No competing interests declared

Funding

National Natural Science Foundation of China (41702002)

  • Jia Jia

National Natural Science Foundation of China (41872008)

  • Ke-Qin Gao

State Key Laboratory of Palaeobiology and Stratigraphy (193111)

  • Jia Jia

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Acknowledgements

We would like to thank C-F Zhou (Shandong University of Science and Technology) for help with field work and stratigraphic data collection; J Anderson (University of Calgary) and J Gardner (Royal Tyrrell Museum) for insightful comments and helpful discussions; J-P Jiang (Chengdu Institute of Biology, [CIB]), A Resetar (Field Museum of Natural History, [FMNH]), C-S Chen (Zhejiang Museum of Natural History), R-C Xiong (Liupanshui Normal University), G Wei (Zunyi Medical University) and S Wang (Capital Normal University) for access to comparative specimens of living salamanders under their curation. We are grateful to Z-X Luo, J Lemberg, A I Neander (all University of Chicago) and M-H Zhang (CIB) for their assistance in CT scanning specimens. We greatly appreciate eLife to generiously waive the publication fee for us, the Senior Editor George Perry, Reviewing Editor and Reviewer Min Zhu and two other Reviewers Pavel Skutschas and David Marjanović for their valuable suggestions. JJ and GL are grateful to J-H JiaLi for her encouragement during the pandemic. JJ is supported by National Natural Science Foundation of China (41702002) and State Key Laboratory of Palaeobiology and Stratigraphy (Nanjing Institute of Geology and Palaeontology, CAS) (193111). K-QG is supported by National Natural Science Foundation of China (41872008).

Senior Editor

  1. George H Perry, Pennsylvania State University, United States

Reviewing Editor

  1. Min Zhu, Institute of Vertebrate Paleontology and Paleoanthropology, Chinese Academy of Sciences, China

Reviewers

  1. Min Zhu, Institute of Vertebrate Paleontology and Paleoanthropology, Chinese Academy of Sciences, China
  2. Pavel Skutschas
  3. David Marjanovic, Museum für Naturkunde, Germany

Publication history

  1. Received: January 7, 2022
  2. Preprint posted: January 17, 2022 (view preprint)
  3. Accepted: May 15, 2022
  4. Accepted Manuscript published: May 16, 2022 (version 1)
  5. Version of Record published: June 6, 2022 (version 2)

Copyright

© 2022, Jia et al.

This article is distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use and redistribution provided that the original author and source are credited.

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  1. Jia Jia
  2. Guangzhao Li
  3. Ke-Qin Gao
(2022)
Palatal morphology predicts the paleobiology of early salamanders
eLife 11:e76864.
https://doi.org/10.7554/eLife.76864

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