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What do ossification sequences tell us about the origin of extant amphibians?

October 2019

DOI:10.1101/352609v3

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Authors:

Michel Laurin

French National Centre for Scientific Research

Oceane Lapauze

Naturhistorisches Museum Basel

David Marjanović

Museum für Naturkunde - Leibniz Institute for Research on Evolution and Biodiversity

The origin of extant amphibians has been studied using several sources of data and methods, including phylogenetic analyses of morphological data, molecular dating, stratigraphic data, and integration of ossification sequence data, but a consensus about their affinities with Paleozoic tetrapods has failed to emerge. We have compiled five datasets to assess the relative support for six competing hypotheses about the origin of extant amphibians: a monophyletic origin among temnospondyls, a monophyletic origin among lepospondyls, a di-phyletic origin among both temnospondyls and lepospondyls, a diphyletic origin among temnospondyls alone, and two variants of a triphyletic origin, in which anurans and urodeles come from different temnospondyl taxa while caecilians come from lepospondyls and are either closer to anurans and urodeles or to amniotes. Our datasets comprise ossification sequences of up to 107 terminal taxa and up to eight cranial bones, and up to 65 terminal taxa and up to seven appendicular bones, respectively. Among extinct taxa, only two or three temnospondyl can be analyzed simultaneously for cranial data, but this is not an insuperable problem because each of the six tested hypotheses implies a different position of temnospondyls and caecilians relative to other sampled taxa. For appendicular data, more extinct taxa can be analyzed, including some lepospondyls and the finned tetrapodomorph Eusthenopteron, in addition to temnospondyls. The data are analyzed through maximum likelihood, and the AICc (corrected Akaike Information Criterion) weights of the six hypotheses allow us to assess their relative support. By an unexpectedly large margin, our analyses of the cranial data support a monophyletic origin among lepospondyls; a monophyletic origin among temnospondyls, the current near-consensus, is a distant second. All other hypotheses are exceedingly unlikely according to our data. Surprisingly, analysis of the appendicular data supports triphyly of extant amphibians within a clade that unites lepospondyls and temno-spondyls, contrary to all phylogenies based on molecular data and recent trees based on paleontological data, but this conclusion is not very robust.

Reference phylogeny used for some of the analyses, illustrating the LH (lepospondyl hypothesis) of lissamphibian origins. The tree was time-calibrated, but analyses showed that branch lengths are irrelevant, given that the best model is speciational (Tables 2-4). Main sources for topology and divergence times: Reeder (2003); Brandley et al. (2005); Pons et al. (2005); Lecompte et al. (2008); Bossuyt and Roelants (2009); Germain and Laurin (2009); Hugall et al. (2007); Gonzales et al. (2009); Meredith et al. (2011); Sterli et al. (2013); Wang et al. (2013); Marjanović and Laurin (2013b, 2019); Pyron (2014); Rabosky et al. (2014); Schoch (2014a); Prum et al. (2015); Zhuang et al. (2015); Tarver et al. (2016); Feng et al. (2017); Irisarri et al. (2017); Lu et al. (2017); Pardo et al. (2017b); Jetz and Pyron (2018). The colored bands represent geological stages from the international geological timescale (Ogg et al. 2016). Several papers, mainly Tarver et al. (2016), were used for the phylogeny and divergence times of mammals. For the Muridae, three references were used: Lecompte et al. (2008), Zhuang et al. (2015), and Lu et al. (2017) for the position of two taxa: Mesocricetus auratus and Peromyscus melanophrys. Other species were placed following the work of Meredith et al. (2011), which also gives divergence times. We caution, however, that all available molecular dates for Paleogene and earlier mammal nodes are controversial and may be overestimates (Berv and Field 2017; Phillips and Fruciano 2018). Three references were also used to integrate squamates in the phylogenetic tree and for the calibration of divergence times: Brandley et al. (2005), Rabosky et al. (2014), Reeder (2003). Sterli et al. (2013) was used for turtles.

…

AICc weights showing relative support for six evolutionary models given various appendicular datasets (3 and 4; see Table 1) and various hypotheses. Because of the number of analyses presented below, only the AICc weights are presented (best values in boldface). Abbreviations: DH, diphyly hypothesis (both versions); LH, lepospondyl hypothesis; TH, temnospondyl hypothesis.

…

Support (AIC and AICc weights) for the six topologies, reflecting the six hypotheses about the origin of extant amphibians, for dataset 2 (see Table 1), which includes seven cranial characters (nasal, parietal, squamosal, premaxilla, maxilla, pterygoid, and exoccipital) and 105 taxa, excluding Sclerocephalus (among Paleozoic taxa, only Apateon is present). Abbreviations, boldface and hypotheses as in Tables 2 and 5.

…

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Preprint uploaded to bioRχiv on 08 October 2019

What do ossification sequences tell us about the origin of

extant amphibians?

Michel Laurin1, Océane Lapauze1, David Marjanović2

1CR2P (Centre de Recherche sur la Paléodiversité et les Paléoenvironments; UMR 7207), CNRS/MNHN/

Sorbonne Université, Muséum national d’Histoire naturelle, Département Histoire de la Terre, 57 rue Cuvier, F-

75231 Paris cedex 05, France; michel.laurin@mnhn.fr

2Museum für Naturkunde (Leibniz Institute for Evolutionary and Biodiversity Research), Invalidenstraße 43, D-

10115 Berlin, Germany; david.marjanovic@gmx.at

ORCID

0000-0003-2974-9835 (Michel Laurin)

0000-0001-9720-7726 (David Marjanović)

ABSTRACT

The origin of extant amphibians has been studied using several sources of data and methods, including

phylogenetic analyses of morphological data, molecular dating, stratigraphic data, and integration of ossification

sequence data, but a consensus about their affinities with Paleozoic tetrapods has failed to emerge. We have

compiled five datasets to assess the relative support for six competing hypotheses about the origin of extant

amphibians: a monophyletic origin among temnospondyls, a monophyletic origin among lepospondyls, a di-

phyletic origin among both temnospondyls and lepospondyls, a diphyletic origin among temnospondyls alone,

and two variants of a triphyletic origin, in which anurans and urodeles come from different temnospondyl taxa

while caecilians come from lepospondyls and are either closer to anurans and urodeles or to amniotes. Our

datasets comprise ossification sequences of up to 107 terminal taxa and up to eight cranial bones, and up to 65

terminal taxa and up to seven appendicular bones, respectively. Among extinct taxa, only two or three

temnospondyl can be analyzed simultaneously for cranial data, but this is not an insuperable problem because

each of the six tested hypotheses implies a different position of temnospondyls and caecilians relative to other

sampled taxa. For appendicular data, more extinct taxa can be analyzed, including some lepospondyls and the

finned tetrapodomorph Eusthenopteron, in addition to temnospondyls. The data are analyzed through maximum

likelihood, and the AICc (corrected Akaike Information Criterion) weights of the six hypotheses allow us to assess

their relative support. By an unexpectedly large margin, our analyses of the cranial data support a monophyletic

origin among lepospondyls; a monophyletic origin among temnospondyls, the current near-consensus, is a

distant second. All other hypotheses are exceedingly unlikely according to our data. Surprisingly, analysis of the

appendicular data supports triphyly of extant amphibians within a clade that unites lepospondyls and temno-

spondyls, contrary to all phylogenies based on molecular data and recent trees based on paleontological data,

but this conclusion is not very robust.

(which was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint. http://dx.doi.org/10.1101/352609doi: bioRxiv preprint first posted online Jun. 20, 2018;

Preprint uploaded to bioRχiv on 08 October 2019

INTRODUCTION

Paleontologists have been studying the origin of

the extant amphibian clades for more than a century.

Early studies generally proposed an origin of at least

some extant amphibians from temnospondyls. Cope

(1888) initially suggested that batrachians (anurans

and urodeles) derived from temnospondyls (a large

clade of limbed vertebrates known from the Early

Carboniferous to the Early Cretaceous) because he

believed that the batrachian vertebral centrum was an

intercentrum, the dominant central element of

temnospondyls. Later, Watson (1940) argued that

anurans were derived from temnospondyls because

of similarities (mostly in the palate) between the

temnospondyl “Miobatrachus” (now considered a

junior synonym of Amphibamus) and anurans. Mono-

phyly of extant amphibians (Lissamphibia) was pro-

posed by Parsons and Williams (1962, 1963), an idea

that was accepted more quickly by herpetologists

than by paleontologists. Lissamphibian monophyly

was supported by (among a few other character

states) the widespread occurrence of pedicellate, bi-

cuspid teeth. The subsequent discovery of such teeth

in the amphibamid temnospondyl Doleserpeton (Bolt

1969) reinforced the widespread acceptance of an

origin of Lissamphibia from within temnospondyls

(e.g., Schoch and Milner 2004). Recently, this hypo-

thesis, referred to as the temnospondyl hypothesis or

TH for short (Fig. 1c), has been supported by several

phylogenetic analyses based on phenotypic data

matrices (e.g. Ruta and Coates 2007; Sigurdsen and

Green 2011; Maddin et al. 2012; Pardo et al. 2017a,

b: fig. S6; Mann et al. 2019).

Other hypotheses about the origin of extant

amphibians have been available in the literature for

nearly as long a time (see Schoch and Milner 2004

for a historical review). These were initially formulated

especially for the urodeles and caecilians, which are

less similar to temnospondyls and lack a tympanic

middle ear (which is present in most anurans and

often inferred for at least some temnospondyls but

absent in lepospondyls). Thus, Steen (1938) highligh-

ted similarities in the palate (broad cultriform process

of the parasphenoid) and cheek (loss of several

bones) between lysorophian lepospondyls and uro-

deles. Carroll and Currie (1975) and Carroll and

Holmes (1980) argued that the exant amphibians had

three distinct origins among early stegocephalians;

while they accepted an origin of anurans among

temnospondyls, they suggested that urodeles and

caecilians originated from two distinct groups of

lepospondyls (Rhynchonkos for caecilians, Hapsido-

pareiidae for urodeles). Later, based mostly on

developmental similarities between the temnospon-

dyl Apateon and urodeles, Carroll (2001, 2007) and

Fröbisch et al. (2007) proposed another hypothesis

involving a triphyletic origin of lissamphibians, with an

origin of anurans and urodeles from two distinct tem-

nospondyl groups, while the caecilians would remain

in the lepospondyl clade. This is what we call the poly-

phyly hypothesis (PH). We have tested two versions.

One (here called PH1; Fig. 1e) was cautiously

suggested by Fröbisch et al. (2007); it agrees with the

paleontological consensus in placing all or most lepo-

spondyls closer to Amniota than to Temnospondyli

(Fig. 1b; Sigurdsen and Green 2011; Pardo et al.

2017a, b: fig. S6; Marjanović and Laurin 2019; Clack

et al. 2019; Mann et al. 2019). The other (PH2; Fig.

1f) is modified to make Lissamphibia monophyletic

with respect to Amniota, a fact we consider

demonstrated beyond reasonable doubt by multiple

phylogenetic analyses of molecular data (Fig. 1a;

Irisarri et al. 2017; Feng et al. 2017; and references

cited therein); this comes at the expense of contra-

dicting the paleontological consensus, which was not

yet established when Milner (1993: 16–18, fig. 5B)

argued for something like the PH2 as one of two more

or less equal possibilities. Anderson (2007) and An-

derson et al. (2008) found lissamphibian diphyly, spe-

cifically a monophyletic, exclusive Batrachia among

the temnospondyls while keeping the caecilians

among the lepospondyls (DH1; Fig. 1g). Pardo et al.

(2017b: fig. 2, S7) presented a similar hypothesis,

with batrachians and caecilians having separate

origins within the temnospondyls (DH2; Fig. 1h); we

should point out, however, that their dataset

contained only temnospondyls and lissamphibians,

and while they found the DH2 using Bayesian infer-

ence, it was only one of four equally parsimonious

results (see Marjanović and Laurin 2019 for this fact

and a discussion of Bayesian analysis of paleontolo-

gical datasets). Further, a monophyletic origin of all

extant amphibians among lepospondyls has also

been proposed (Laurin 1998; Pawley 2006: appendix

16; Marjanović and Laurin 2009, 2013a, 2019). This

will be referred to below as the lepospondyl hypo-

thesis (LH; Fig. 1d).

Phylogenetic analyses of molecular data cannot

distinguish the TH, the PH2, the DH2 or the LH from

each other by topology (Fig. 1) because all of these

imply lissamphibian monophyly with respect to

amniotes, and molecular data are not available from

any other tetrapodomorphs. Several other types of

data and methods have, however, been used to try to

discriminate between the various hypotheses on the

origin of extant amphibians. In addition to classical

phylogenetic analyses of morphological data matri-

ces, these include the use of molecular dating (Zhang

et al. 2005; Marjanović and Laurin 2007; Pardo et al.

2017b) and stratigraphic data (Marjanović and Laurin

2008) to compare the inferred divergence dates be-

tween the three main extant amphibian clades on the

basis of molecular data with predictions based on the

fossil record under the TH and the LH on one side

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Laurin et al. 2019 — Lissam phibian origin and ossification sequences

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and the PH and the DH on the other. However,

developmental data, in the form of ossification

sequences, have been the second-most frequently

used (after classic morphological data) to argue for

particular phylogenetic hypotheses. These data

include mainly cranial (e.g. Schoch 2002, 2006;

Schoch and Carroll 2003; Schoch and Milner 2004;

Anderson 2007; Carroll 2007; Germain and Laurin

2009) and autopodial ossification sequences (e.g.

Fröbisch et al. 2007, 2015). Ossification sequences

of other parts of the skeleton, like the vertebrae,

shoulder girdle and scales, are also documented in a

few Paleozoic stegocephalians (e.g. Carroll et al.

1999; Witzmann and Schoch 2006; Anderson 2007;

Carroll 2007; Olori 2013), not to mention finned

tetrapodomorphs (Cloutier 2009), but these have

played a minor role in the controversy about the origin

of extant amphibians. Recently, Danto et al. (2019)

concluded that vertebral ossification sequences

varied too quickly and could not be used to assess

the origin of lissamphibians. This study relies on both

cranial and appendicular ossification sequences and

compares their implications for tetrapod phylogeny.

MATERIAL AND METHODS

Ossification sequence data

From all the literature we could access, we

compiled the most extensive database on ossification

sequences for osteichthyans that exists to date. The

most useful sources for extant taxa included

compilations: Harrington et al. (2013) for amphibians,

Weisbecker and Mitgutsch (2010) for anurans, Hugi

et al. (2012) for squamates, Maxwell et al. (2010) for

birds, and Koyabu et al. (2014) and Weisbecker

(2011) for mammals. The cranial and appendicular

sequences of Permian temnospondyls (the stereo-

spondylomorphs Sclerocephalus and Archegosau-

rus, the non-branchiosaurid “branchiosaur” Micromel-

erpeton and the branchiosaurids “Melanerpeton”

humbergense, Apateon caducus and A. pedestris)

were assembled from several references cited in the

Appendix; note that the two Apateon species are

each represented by two different sequences scored

after populations from two separate paleo-lakes

(Erdesbach and Obermoschel) in which both species

occur. Appendicular ossification sequences of the

lepospondyls Microbrachis and Hyloplesion are in-

corporated from Olori (2013), that for the finned

tetrapodomorph Eusthenopteron was combined from

Cote et al. (2002) and Leblanc and Cloutier (2005).

All sources of our sequence data can be found in

the Appendix. The sequences themselves and the

phylogenetic trees corresponding to the tested

hypotheses are included in the supplements, which

are posted on the bioRχiv page from which this paper

is available. The sequences were not used to

generate the tree topology or the branch lengths

(which represent evolutionary time); the tree is

compiled from published sources (provided below)

which did not use any ossification sequences in their

phylogenetic analyses.

Figure 1 (next page). Hypotheses on the relationships of the extant amphibian clades since the late 20th century. The names

of terminal taxa sampled here for cranial characters are in boldface, those sampled for appendicular characters are

underlined; the names of larger clades are placed toward the right end of a branch if they have minimum-clade (node-based)

definitions, to the left if they have maximum-clade (branch-based) definitions. Names in parentheses would, given that

phylogenetic hypothesis, not be used, but replaced by synonyms. Among terminal taxa, “Melanerpeton” humbergense,

sampled for appendicular characters, is not shown, but is always the sister-group of Apateon; Microbrachis, likewise sampled

for appendicular characters, is not shown either, but is always the sister-group of Hyloplesion; Eusthenopteron is not shown

in c)–h), where it forms the outgroup (b)). See text for Micromelerpeton and for references. The first two trees (a, b) show

the current consensus; the other trees (c–h) show the various tested paleontological hypotheses. Abbreviations: D.,

Dissorophoidea; S., Stereospondylomorpha. a) Consensus of the latest phylogenetic analyses of molecular ; all named

clades are therefore extant. Note the monophyly of the extant amphibians (Lissamphibia, marked with a light gray dot) with

respect to Amniota. b) Consensus of all analyses of Paleozoic limbed vertebrates, omitting the extant amphibian clades.

Note the monophyly of “lepospondyls” + amniotes (marked with a dark gray dot). c) TH: “temnospondyl hypothesis”. Lissam-

phibia nested among dissorophoid temnospondyls. Compatible with both a) and b) (gray dots). d) LH: “lepospondyl hypothe-

sis”. Lissamphibia nested among “lepospondyls”; consequently, temnospondyls are not crown-group tetrapods. Compatible

with both a) and b) (gray dots). e) PH1: “polyphyly hypothesis”, first variant. Urodela as dissorophoid temnospondyls close

to Apateon, Anura as a separate clade of dissorophoid temnospondyls, Gymnophiona as “lepospondyls”. Compatible with

b) (dark gray dot) but not with a) (light gray circle). f) PH2: “polyphyly hypothesis”, second variant. Like PH1, but with restored

monophyly of extant amphibians with respect to amniotes (light gray dot; see a)) at the expense of compatibility with the

paleontological consensus concerning the position of temnospondyls, lepospondyls, and amniotes (dark gray circle; see b)).

g) DH1: “diphyly hypothesis”, first variant. Batrachia as dissorophoid temnospondyls, Gymnophiona as “lepospondyls”.

Compatible with b) (dark gray dot) but not with a) (light gray circle). h) DH2: “diphyly hypothesis”, second variant. Batrachia

as dissorophoid temnospondyls, Gymnophiona as stereospondylomorph temnospondyls. Compatible with both a) and b).

(which was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.

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Laurin et al. 2019 — Lissam phibian origin and ossification sequences

PREPRINT

Apateon

Sclerocephalus

Anura

Urodela

Gymnophiona

Amniota

Apateon

Hyloplesion

Sclerocephalus

Anura

Urodela

Gymnophiona

Amniota

Apateon

Anura

Urodela

Gymnophiona

Amniota

Compatibility:

Lissamphibia

Lepospondyli + Amniota

Lissamphibia

Compatibility:

molecular consensus paleontological consensus

PH1

Compatibility:

PH2

Compatibility:

DH1

Compatibility:

DH2

Compatibility:

Lissamphibia

Lepospondyli + Amniota

Lissamphibia

not Lepospondyli + Amniota

Lissamphibia

Lepospondyli + Amniota

Latimeria

Dipnoi

Anura

Urodela

Gymnophiona

Amniota

Lissamphibia

(Lissamphibia)

Batrachia

Lepospondyli

Dipnomorpha

Tetrapoda

Sarco-

pterygii

Sarco-

pterygii

Tetrapodo-

morpha

Tetrapodo-

morpha

Actinistia

Amphibia

Actinistia

Dipnomorpha

Apateon

Sclerocephalus

Temno-

spondyli

Sclerocephalus

Temnospondyli

Amphibia

(Temnospondyli)

Amphibia

(Temnospondyli)

Apateon

Sclerocephalus

Hyloplesion

Anura

Urodela

Gymnophiona

Amniota

(Lissamphibia)

(Batrachia)

Tetrapoda

Temnospondyli

Lepospondyli

Hyloplesion

Micromelerpeton

Hyloplesion

Micromelerpeton

Lepospondyli

Stereospondylomorpha D.

Dissorophoidea

(Lepospondyli)

Ichthyostega

Eusthenopteron

Amniota

a) b)

c) d)

e) f)

g) h)

Apateon

Hyloplesion

Sclerocephalus

Anura

Urodela

Gymnophiona

Amniota

not Lissamphibia

Lepospondyli + Amniota

Batrachia

Tetrapoda

Temnospondyli

Lepospondyli

Apateon

Sclerocephalus

Hyloplesion

Anura

Urodela

Gymnophiona

Amniota

not Lissamphibia

Lepospondyli + Amniota

(Batrachia)

Tetrapoda

Temnospondyli

Lepospondyli

Lepospondyli Hyloplesion

Archegosaurus

Archegosaurus Archegosaurus

Archegosaurus

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Preprint uploaded to bioRχiv on 08 October 2019

The software we used to compute AICc weights,

the CoMET module (Lee et al. 2006) for Mesquite 3.6

(Maddison and Maddison 2018), cannot handle

missing data. This unfortunately meant we had to

discard much information. In order to keep as many

taxa as possible in the analysis, we first compiled a

matrix (not shown) of 244 taxa and 213 characters.

All of these characters are positions of skeletal elem-

ents (cranial, appendicular, axial and others) in ossifi-

cation sequences, standardized between 0 and 1

following Germain and Laurin (2009), as explained

below. Of these, we kept characters that were scored

in the Paleozoic taxa in our initial database, and ex-

tant taxa that were scored for the same sets of

characters. This resulted in two initial datasets, one

of cranial and one of appendicular sequences (it was

not possible to include both sets of sequences to-

gether because this would have left too few taxa in

the matrix).

In the end, however, we were left with three over-

lapping cranial datasets. The largest cranial dataset

we could make, dataset 2 of Table 1, has 105 taxa

(103 extant, plus the two species of Apateon scored

from Erdesbach) and seven characters: the appear-

ance times of the premaxilla, maxilla, nasal, parietal,

pterygoid, exoccipital and squamosal bones. It lacks

Sclerocephalus, which cannot be scored for the ap-

pearance time of the squamosal. This is unfortunate

because Sclerocephalus is one of only three extinct

taxa for which a usable cranial ossification sequence

is known at all, and further because it occupies a

special place in the DH2, according to which it lies on

the caecilian stem. We attempted to compensate for

this deficiency by assembling two more cranial data-

sets: dataset 1, which contains 107 taxa (104 extant,

Apateon spp. from Erdesbach, and Sclerocephalus)

but only six characters by lacking the squamosal, and

dataset 5, which includes 84 taxa (81 extant, Apateon

spp. from Erdesbach, and Sclerocephalus) and eight

cranial characters (the vomer and the frontal bone

are added to the six of dataset 1).

For the appendicular characters, in addition to

dataset 3 which contains seven characters (humerus,

radius, ulna, ilium, femur, tibia and fibula) and 62 taxa

(54 extant, Apateon spp. from Obermoschel, Sclero-

cephalus, Archegosaurus, Micromelerpeton, Hylople-

sion, Microbrachis and Eusthenopteron), another

(dataset 4) includes only four characters (radius, ulna,

ilium, and femur), but it features 65 sequences, the

additional data being Apateon spp. from Erdesbach

and “Melanerpeton” humbergense. See Table 1 for a

list of these datasets and the supplements for the

datasets themselves.

The data loss in these various datasets is not as

severe as it may first seem, because most of the

characters that have been excluded from these analy-

ses had less than 10% scored cells (sometimes less

than 1%), and most of them could not be scored for

any temnospondyl or lepospondyl, so they could not

have helped resolve the main question examined in

this study.

The order in which the sampled cranial bones

ossify varies substantially in our sample of taxa, but

based on simple (not phylogenetically-weighted)

average position, the frontal appears first, followed

closely by the premaxilla, parietal, and maxilla (in

close succession), and then by the squamosal, ex-

occipital, pterygoid, and last by the nasal. However,

all of these bones ossify first (among these bones; not

necessarily in the whole skeleton) in at least one of

the included taxa. Among the appendicular bones,

there is more variability; all ossify first in at least one

of the 62 sampled taxa, and three (radius, ulna and

ilium) ossify last in at least one taxon.

Due to the homology problems between the skull

bones of tetrapods and actinopterygians and missing

data, we had to omit all actinopterygians from our

analyses. As cranial ossification sequences remain

poorly documented for extant finned sarcopterygians,

except perhaps lungfish, whose skull bones seem

mostly impossible to homologize (Criswell 2015), our

analyses of those data are restricted to limbed

vertebrates. However, for appendicular data, we were

able to include the Devonian tristichopterid Eustheno-

pteron foordi.

Unfortunately, the only cranial ossification

sequence available for any supposed lepospondyl,

that of the aïstopod Phlegethontia longissima, is

documented from only three ossification stages

(Anderson et al. 2003; Anderson 2007). This poses a

problem for our analysis method, which assumes that

character evolution can be modeled as Brownian

motion; this assumption is decreasingly realistic as

the number of character states (sequence positions)

decreases, because the resulting distribution devi-

ates increasingly from that of a continuous character.

Furthermore, some recent anatomical restudies and

phylogenetic analyses suggest that aïstopods are not

lepospondyls, but early-branching stem-stegocepha-

lians (Pardo et al. 2017a, 2018; Mann et al. 2019;

Clack et al. 2019).

The low taxon sample is more limiting for this

analysis than the low character sample. However, as

explained below, the absence of lepospondyl

sequences in our cranial dataset does not preclude

testing the six hypotheses (TH, PH1, PH2, DH1, DH2,

LH; see above or Figure 1 for the explanation of these

abbreviations) because each of these six hypotheses

makes different predictions about where temnospon-

dyls and caecilians fit relative to other taxa. Thus, in

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the absence of lepospondyls in our dataset, the tests

of these hypotheses are somewhat indirect and

inference-based, but they remain possible. Our tests

based on appendicular data include two lepospondyls

(Hyloplesion longicostatum and Microbrachis pelika-

ni), but the absence of caecilians in that dataset

proves more limiting than the absence of lepospon-

dyls in the cranial dataset because the TH, DH1 and

DH2 become indistinguishable (Fig. 1c, g, h). How-

ever, the presence of the temnospondyl Micromel-

erpeton allows us to test two variants of the TH/DH

distinguished by the monophyly (e.g. Ruta and

Coates 2007) or polyphyly (e.g. Schoch 2018) of

“branchiosaurs” (the temnospondyls Apateon,

“Melanerpeton” humbergense and Micromelerpeton).

Table 1. List of datasets used in this paper. All are subsets of our global compilation that were selected to meet the

requirement of the method used (missing data cannot be handled). The temnospondyl species Apateon caducus and A.

pedestris are included in all datasets, but scored after populations from two different paleo-lakes in which both species occur.

Dataset number

Type of characters

cranial

appendicular

cranial

Number of

characters

Number of taxa

107

105

Sclerocephalus

yes

Source of data for

Apateon

Erdesbach

Obermoschel

Erdesbach and

Obermoschel

Erdesbach

Additional

Paleozoic taxa

None

Archegosaurus,

Micromelerpeton,

Hyloplesion,

Microbrachis,

Eusthenopteron

Archegosaurus,

Micromelerpeton,

“Melanerpeton”

humbergense,

Hyloplesion,

Microbrachis,

Eusthenopteron

None

Table in which it is

used

2, 5

3, 6

4, 8

4, 9

Sensitivity analysis for sequence

polymorphism

Given the potential impact of intraspecific

variability in ossification sequence on inferred nodal

sequences and heterochrony (Olori 2013; Sheil et al.

2014), we compiled two consensus sequences for

Apateon caducus and A. pedestris each, represent-

ing two localities where both species occur, the paleo-

lakes of Erdesbach (Schoch 2004) and Obermoschel

(Werneburg 2018). Based on dataset 4 (see Table 1),

we incorporated these into a global and two separate

analyses (one analysis per locality) to determine the

impact of the observed variability. As detailed above,

incorporating the sequences from Erdesbach re-

duced the number of characters from seven to only

four because the software used cannot handle mis-

sing data (see above and below), but this information

loss is compensated by the great increase in number

of sequences from extinct taxa (eleven instead of two,

when counting the sequences of Apateon from both

localities separately) and the fact that this includes

some lepospondyls (see below). It would have been

even better to perform a sensitivity analysis

incorporating variability for all taxa for which such

information was available, but given the scope and

nature of our study, this would have been exceedingly

time-consuming and is best left for the future.

Standardization of the data

Given that various taxa differ in their numbers of

bones and that the resolution of the sequences is also

variable between taxa, these data needed to be

standardized to make comparisons and computations

meaningful, as suggested by Germain and Laurin

(2009). Note that we performed this standardization

on the complete dataset of characters, before filtering

for data completeness. This complete dataset (not

shown) includes 213 cranial, appendicular and other

characters, but no taxon is scored for all characters,

because that matrix has much missing data. For

instance, the most completely scored taxon, Amia

calva, still has 57.4% missing data (more than half),

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which indicates that 92 characters were scored for

this taxon, including several ties (the resolution was

41 positions, so they varied by increments of 0.025 or

2.5% of the recorded ontogeny). We did not re-stan-

dardize after filtering characters out because we be-

lieve that the initial standardization better reflects the

relative position of events in development than a stan-

dardization based on only seven events in ontogeny.

Because of this, some characters in the reduced da-

tasets lack states 0 or 1 for some taxa. This is simply

because the first or last events in the ontogenetic

sequence were filtered out. Thus, we used the posi-

tion in the sequence (from first to last, in the complete

dataset) and standardized this relative sequence po-

sition between 0 and 1 using the formula given by

Germain and Laurin (2009). The standardized se-

quence position (Xs) is:

Xs = (Xi – Xmin)/(Xmax – Xmin),

where:

Xi is the position of a given bone in the sequence

Xmin is the lowest position in the sequence

(generally denoted 0 or 1)

Xmax is the highest position in the sequence (for

instance, if there are 20 bones, Xmin is 1 and the

sequence is completely resolved, Xmax = 20).

This yields a standardized scale that varies

between 0 and 1 for each taxon, in which 0 and 1 are

the positions of the first and last events in the

sequence, respectively. For instance, for Ambystoma

maculatum (an extant urodele), in the original data-

set, the first events (tied) were the ossification of pre-

maxilla, vomer, dentary and coronoid (standardized

position: 0); the last event was the articular (standard-

ized position: 1), and there is a resolution of 12 posi-

tions (hence, increments of 0.0909 or 1/11). However,

in the final dataset of 7 charcters, the articular is ab-

sent; hence, the first bone in the sequence is the pre-

maxilla, at a standardized position of 0, and the last

is the nasal, as a standardized position of 0.8181 be-

cause all events in position 1 (articular) and 0.9091

(stapes) have been filtered out.

We also experimented with using size (skull

length) or developmental stage as standards, but this

led to lower sequence resolution because body size

is not available for all sequence positions and for all

taxa (results not shown), so we worked only with se-

quences standardized by position. Given that our

data filtering procedure retains few data (only six,

seven or eight characters for the cranial dataset, and

four or seven characters for the postcranial dataset),

it is important to use the method that discards the

least amount of data, and this was achieved by using

sequence position. We do not imply that standardiz-

ing by size is not recommended in general. On the

contrary, if good body size data were available for all

taxa and all developmental stages, this should be a

better strategy, and only having access to absolute

time should be even better. However, practical

limitations of data availability prevent us from using

these methods now.

Our ossification sequence data (reduced dataset

of four to eight characters) of extant and extinct taxa,

and the phylogenetic trees we used, are available in

the supplements.

Analysis methods

To discriminate between the six hypotheses about

the origin of extant amphibians, two methods are

available: direct phylogenetic analysis of the se-

quence data, and comparisons of the tree length

(number of steps in regular parsimony, squared

length in squared-change parsimony, likelihood, or

similar measures) of various trees selected a priori to

represent these hypotheses (in these trees, only the

position of caecilians and extinct taxa, here temno-

spondyls and lepospondyls, varies). We used both

approaches but expected the second to perform

much better because relatively few data are available,

and thus, phylogenetic analysis of such data is

unlikely to provide a well-resolved tree.

For the first approach, we first transformed the

standardized sequence positions back into discrete

characters using formulae in a spreadsheet and

scaled the characters so that the highest state in all

would be 9. This ensures that each character has

equal weight in the analysis, regardless of its varia-

bility in the ossification sequence. The characters

were ordered to reflect the assumed evolutionary

model (ontogenetic timing is a quantitative character

that was discretized) and because for such charac-

ters, ordering yields better results (Rineau et al. 2015,

2017; see discussion in Marjanović & Laurin 2019).

The resulting data matrices (one for cranial and

another for appendicular characters, both with seven

characters each) were analysed using parsimony in

PAUP* 4.0a165 (Swofford 2019). We used the TBR

(tree bisection-reconnection) branch swapping

algorithm and performed a search with 50 random

addition replicates (or several such searches, for the

cranial data) while holding two trees at each step and

with a maximum number of trees set at one million.

For cranial data, the main search lasted about 100

hours on a MacBook Pro Retina with a 2.5 GHz iCore

7 quadri-core processor and 16 GB RAM. The exact

search time cannot be reported because PAUP*

crashed after saving the trees to a file for one of the

longest runs (several analyses were made, over

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several days), but before the log could be saved. The

analysis of the seven appendicular characters was

much faster (27 minutes and a half), presumably

because that matrix has fewer taxa (62 instead of

105).

For the second approach (comparison of fit of

various trees selected a priori to reflect previously

published hypotheses), we used the CoMET module

(Lee et al. 2006) for Mesquite 3.6 (Maddison and

Maddison 2018) to test the relative fit of the data on

trees representing the six hypotheses. CoMET calcu-

lates the likelihood and the AIC (Akaike Information

Criterion) of nine evolutionary models given continu-

ous data and a tree. Note that our data only represent

an approximation of continuous data; if standardiza-

tion had been performed on developmental time or

body size, the data would actually have been continu-

ous. Standardization was carried out using sequence

position because of data limitation problems, so the

data actually follow a decimalized meristic scale.

However, the difference between these situations de-

creases as the number of sequence positions increa-

ses, and our global scale includes up to 41 positions

(and an average of 10.9 positions), so our data should

approximate a continuous distribution sufficiently well

for our analyses to be valid. This consideration pre-

vents us from adding the highly apomorphic aïstopod

Phlegethontia, for which only three cranial ossifica-

tion stages are known (Anderson et al. 2003; Ander-

son 2007); moreover, five of the seven bones inclu-

ded in our analyses appear in the last two of these

stages, and two of the relevant bones (parietal and

exoccipital) are not present as separate ossifications,

which would create additional missing data. In that

case, the very low number of stages would create

strong departures from the assumption of continuous

data. This would probably create statistical artifacts,

and the uncertainty about the position of Phlegethon-

tia (Pardo et al. 2017a, 2018; Marjanović and Laurin

2019; Clack et al. 2019) would complicate interpreta-

tion of the results.

The nine models evaluated by CoMET are

obtained by modifying the branch lengths of the

reference tree. Thus, branches can be set to 0 (for

internal branches only, to yield a non-phylogenetic

model), to 1 (equal or speciational model), left un-

changed from their original length (gradual evolution

in our case, where the original lengths represent geo-

logic time), or set free and evaluated from the data

(free model). This can be applied to internal and/or

external branches, and various combinations of these

yield nine models (Lee et al. 2006: fig. 1). Among

these nine models two have been frequently dis-

cussed in the literature and are especially relevant.

The first is gradual evolution, in which branch lengths

(here representing evolutionary time) have not been

changed. The second is the speciational model, in

which all branches are set to the same length

because changes are thought to occur at speciation

events, which are typically equated with cladogene-

ses in evolutionary models (Bokma et al. 2016).This

model has some similarities with Eldredge and

Gould’s (1972) punctuated equilibria (though a model

with one internal branch stemming from each node

set to 0 and the other set to 1 would be even closer

to the original formulation of that model). In this study,

we assessed the fit of six of the nine models covered

by CoMET; the other three (the punctuated versions

of distance [original branch length], equal and free) in

which the one of each pair of daughter-lineages has

a branch length of zero, could not be assessed due

to problems in the current version of CoMET and

possibly the size of our dataset.

Provided that the same evolutionary model is

optimal for all compared phylogenetic hypotheses

(this condition is met, as shown below), the AIC

weights of the various trees under that model can be

used to assess the support for each tree. In such

comparisons, the topology is part of the evolutionary

model, and the data are the sequences. These com-

parisons can show not only which tree is best sup-

ported, but how many times more probable the best

tree is compared to the alternatives. This quantifica-

tion is another reason to prefer this approach over a

phylogenetic analysis (performed below, but with the

poor results that we anticipated), which can at best

yield a set of trees showing where the extinct taxa

most parsimoniously fit (if we had dozens of charac-

ters, this might be feasible). Comparisons with other

hypotheses through direct phylogenetic analysis are

not possible. Given the small sample size (which here

is the number of characters), we computed the cor-

rected AIC (AICc) and the AICc weights using the

formulae given by Anderson and Burnham (2002) and

Wagenmakers and Farrell (2004).

Our tests make sense only in the presence of a

phylogenetic signal in the data. In addition to the test

of evolutionary model in CoMET mentioned above

(which tests non-phylogenetic as well as phylogenetic

models), we performed a test based on squared-

change parsimony (Maddison 1991) and random

taxon reshuffling (Laurin 2004). For this test, we com-

pared the length of the LH (lepospondyl hypothesis;

Fig. 1d) reference tree (with and without Sclero-

cephalus) to a population of 10,000 random trees

produced by taxon reshuffling.

It could be argued that using other methods (in

addition to the method outlined above) would have

facilitated comparisons with previous studies. How-

ever, the two main alternative methods, event-pair

cracking with Parsimov (Jeffery et al. 2005) and

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Parsimov-based genetic inference (PGI; Harrison

and Larsson 2008), have drawbacks that led us to not

using them. Our objections against event-pair

cracking with Parsimov were detailed by Germain and

Laurin (2009). In short, that method requires an un-

necessary decomposition of sequences into event

pairs, and it cannot incorporate absolute timing infor-

mation (in the form of time, developmental stage or

body size, for instance) or branch length information.

More importantly, the simulations performed by Ger-

main and Laurin (2009) showed that event-pair crack-

ing with Parsimov yields more artefactual change and

has lower power to detect real sequence shifts. That

method is also problematic when trying to infer ances-

tral sequences and can lead to impossible ancestral

reconstructions (e.g. A occurs before B, B occurs

before C, and C occurs before A), as had been docu-

mented previously (Schulmeister and Wheeler 2004:

55). This would create problems when trying to com-

pare the fit of the data on various phylogenetic hypo-

theses. The performance of Parsimov-based genetic

inference (PGI; Harrison and Larsson 2008) has not

been assessed by simulations, but it rests on an edit

cost function that is contrary to our working hypothe-

sis (that the timing of developmental events can be

modeled with a bounded Brownian motion model,

which is assumed by continuous analysis). More spe-

cifically, Harrison and Larsson (2008: 380) stated that

their function attempts to minimize the number of

sequence changes, regardless of the magnitude of

these changes. We believe that disregarding the size

of changes is unrealistic, as shown by the fact that

Poe’s (2006) analyses of thirteen empirical datasets

rejected that model (which he called UC, for

unconstrained change) in favor of the model we

accept (AJ for adjacent states, which favors small

changes over large ones). Furthermore, analyses of

ossification sequence data using techniques for

continuous data as done here (see above) have been

performed by an increasingly large number of studies

(e.g., Skawiński and Borczyk 2017; Spiekman and

Werneburg 2017; Werneburg and Geiger 2017, just

to mention papers published in 2017), so the issue of

ease of comparisons of our results with other studies

is not as serious as it would have been only a few

years ago, and it should be decreasingly so in the

future.

Reference phylogenies

We built a reference timetree that attempts to

capture the established consensus (Fig. 2; see the

next paragraphs for the sources). The tree was

compiled in Mesquite versions up to 3.6 (Maddison

and Maddison 2018) and time-calibrated using the

Stratigraphic Tools module for Mesquite (Josse et al.

2006). For consistency and to avoid the effects of

gaps in the fossil record, we used molecular diver-

gence dates whenever possible. The tree had to be

time-scaled because many of the evolutionary

models that we fit on the tree in the first series of tests

(to determine which evolutionary model can be used

to compare the fit of the hypotheses) use branch

lengths to assess model fit. Note that our procedure

requires estimating divergence times between all

taxa (geological ages of all nodes). When taxa are

pruned, branch lengths are adjusted automatically.

The main sources we used for topology and diver-

gence times (and hence branch lengths) are as

follows:

The phylogeny of lissamphibians follows the work

of Jetz and Pyron (2018). However, several other

sources have been used for the temporal calibration

of the tree: Germain and Laurin (2009) was used for

the urodeles, whereas Feng et al. (2017), supplemen-

ted by Bossuyt and Roelants (2009) and Pyron

(2014), was used for the anurans as well as more

rootward nodes (Batrachia, Lissamphibia, Tetrapoda;

also Amniota). Marjanović and Laurin (2013b) was

used for the Ranidae, Ceratophryidae and Hylidae.

The sediments that have preserved the

temnospondyls Apateon and Sclerocephalus are not

easy to correlate with each other or with the global

chronostratigraphic scale. Combining stratigraphic

information from Schoch (2014a), Schneider et al.

(2015) and Werneburg (2018), we have placed all

three sampled species (A. pedestris, A. caducus, S.

haeuseri) at the Sakmarian/Artinskian stage bounda-

ry (Permian; 290.1 Ma ago); combining stratigraphic

information from Schneider et al. (2015) with the

phylogeny in Schoch (2014a), we have tentatively

placed the divergence between the two Apateon spe-

cies (which are not sister-groups: Schoch 2014a) at

the Kasimovian/Gzhelian stage boundary (Carboni-

ferous; 303.7 Ma ago). The age of the last common

ancestor of Apateon and Sclerocephalus depends

strongly on temnospondyl phylogeny, which remains

unresolved (Pardo et al. 2017b; Marjanović and Lau-

rin 2019; and numerous references in both); as a

compromise between the various options, we have

provisionally placed it at the boundary between the

Early and the Late Carboniferous (Serpukhovian/

Bashkirian, 323.2 Ma ago) where applicable.

We sampled many extant amniotes to achieve

broad coverage of Tetrapoda. For the birds, Pons et

al. (2005) was used for the Laridae, Wang et al.

(2013) for the Phasianidae and Gonzales et al. (2009)

for the Anatidae. The temporal calibration was taken

from Prum et al. (2015) as recommended by Berv

and Field (2017); gaps were filled in using the

database www.birdtree.org.

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Figure 2. Reference phylogeny used for some of the analyses, illustrating the LH (lepospondyl hypothesis) of lissamphibian

origins. The tree was time-calibrated, but analyses showed that branch lengths are irrelevant, given that the best model is

speciational (Tables 2–4). Main sources for topology and divergence times: Reeder (2003); Brandley et al. (2005); Pons et

al. (2005); Lecompte et al. (2008); Bossuyt and Roelants (2009); Germain and Laurin (2009); Hugall et al. (2007); Gonzales

et al. (2009); Meredith et al. (2011); Sterli et al. (2013); Wang et al. (2013); Marjanović and Laurin (2013b, 2019); Pyron

(2014); Rabosky et al. (2014); Schoch (2014a); Prum et al. (2015); Zhuang et al. (2015); Tarver et al. (2016); Feng et al.

(2017); Irisarri et al. (2017); Lu et al. (2017); Pardo et al. (2017b); Jetz and Pyron (2018). The colored bands represent

geological stages from the international geological timescale (Ogg et al. 2016).

Several papers, mainly Tarver et al. (2016), were

used for the phylogeny and divergence times of

mammals. For the Muridae, three references were

used: Lecompte et al. (2008), Zhuang et al. (2015),

and Lu et al. (2017) for the position of two taxa:

Mesocricetus auratus and Peromyscus melanophrys.

Other species were placed following the work of

Meredith et al. (2011), which also gives divergence

times. We caution, however, that all available molecu-

lar dates for Paleogene and earlier mammal nodes

are controversial and may be overestimates (Berv

and Field 2017; Phillips and Fruciano 2018).

Three references were also used to integrate

squamates in the phylogenetic tree and for the

calibration of divergence times: Brandley et al.

(2005), Rabosky et al. (2014), Reeder (2003). Sterli

et al. (2013) was used for turtles.

For turtles, there is now a near-consensus that

they are diapsids, a hypothesis that is not necessarily

incompatible with an origin among “parareptiles”

(Laurin and Piñeiro 2017). Thus, following most re-

cent phylogenetic analyses of molecular data (e.g.,

Hugall et al. 2007; Irisarri et al. 2017), we have in-

serted them as the sister-group of Archosauria.

We disagree with several of the calibration dates

in Irisarri et al. (2017), which often appear unreason-

ably old. For instance, they place the divergence

between caecilians and batrachians and the diver-

gence between anurans and urodeles in the Early

Carboniferous, around 330 and 320 Ma, respectively,

but our thorough analyses of the fossil record, with

due consideration of its incompleteness, suggest

significantly more recent dates, in the Permian

(Marjanović and Laurin 2007, 2008, 2013b). This is

not surprising because some of the dating constraints

used by Irisarri et al. (2017: table S8) are wrong. For

instance, they enforced a minimal divergence age

between cryptodiran and pleurodiran turtles of 210

Ma (Late Triassic), but all analyses of the last fifteen

years (e.g. Sterli et al. 2013, 2018) strongly suggest

that the oldest known turtles that fit within this

dichotomy date from the Late Jurassic, less than 165

Ma. The divergence between humans and armadillos

(boreotherian and xenarthran placentals) was con-

strained to the middle of the Cretaceous (95.3–113

Ma), based on outdated literature that assigned a

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wide variety of stem-eutherians to highly nested posi-

tions in the placental crown; there are currently no

clear placentals known from any Cretaceous sedi-

ments even as young as 66 Ma (see e.g. Halliday et

al. 2015, 2016; Davies et al. 2017; Phillips and Fru-

ciano 2018), barely half the age of the older end of

the constraint range. Conversely, the divergence be-

tween diapsids (hence sauropsids) and synapsids

had a minimal age constraint of 288 Ma (Early

Permian), which is much too young given the pres-

ence of sauropsids (and presumed synapsids) in Jog-

gins, in sediments that have recently been dated

(Carpenter 2015) around 317–319 Ma (early Late

Carboniferous). Thus, we have not used divergence

dates from that source.

To discriminate among the hypotheses on lissam-

phibian origins, we inserted the temnospondyl Apate-

on in the tree where each predicts that it should be

(Fig. 1c–h). Thus, according to the TH (temnospondyl

hypothesis; Fig. 1c), Apateon lies on the lissamphibi-

an stem. Under the LH (lepospondyl hypothesis; Fig.

1d), Apateon lies on the tetrapod stem. Under both

versions of the DH (diphyly hypothesis; Fig. 1g, h),

Apateon lies on the batrachian stem. Under both

versions of the PH (polyphyly hypothesis; Fig. 1e, f),

Apateon lies on the caudate stem. Within the DH and

the PH, both versions of each differ in the position of

Gymnophiona. Thus, despite the absence of any

lepospondyl in our cranial ossification sequence

datasets, our taxonomic sample allows us to test all

these competing hypotheses. The appendicular data-

sets allow more direct tests of some of these hypo-

theses because they include two lepospondyl taxa,

which were likewise placed in trees representing the

tested hypotheses (Fig. 1).

Sclerocephalus is the sister-group of Apateon

under the LH (Fig. 1d), immediately rootward of it (on

the lissamphibian stem) under the TH (Fig. 1c) and

likewise (but on the batrachian stem) under the DH1

(Fig. 1g), on the caecilian stem under the DH2 (Fig.

1h) and the sister-group of Batrachia (including

Apateon) under both versions of the PH (Fig. 1e, f).

“Melanerpeton” humbergense (appendicular data

only) is the sister-group of Apateon in all trees, except

under the hypothesis of branchiosaur paraphyly;

Eusthenopteron (appendicular data only) forms the

outgroup in all trees.

The lepospondyls Microbrachis and Hyloplesion,

from both of which only appendicular data are

available, form an exclusive clade (Marjanović and

Laurin 2019; Clack 2019). This clade is the sister-

group of Lissamphibia (represented only by Batra-

chia) under the LH (because caecilians are lacking

from the appendicular datasets), of Amniota under

the TH and both versions of the DH (these three can-

not be distinguished due to the absence of caecilians)

as well as under the PH1, and of Temnospondyli

(including Batrachia) under the PH2 (see the legend

of Figure 1 for an explanation of these abbreviations).

The temnospondyl Micromelerpeton, from which

likewise only appendicular data are available, forms

the sister-group of Apateon under the LH. The uncer-

tainty over its phylogenetic position within Dissoroph-

oidea (as the sister-group to the rest, including an-

urans and urodeles: e.g. Schoch 2018; as the sister-

group of Apateon + “Melanerpeton” humbergense:

e.g. Ruta & Coates 2007; Marjanović and Laurin

2019) generates two versions of the TH/DH1/DH2

tree for the appendicular dataset. We tested both of

these versions against that dataset, for a total of five

trees.

To ensure that our analyses were not biased in

favor of a given hypothesis, and in case that a contin-

uous evolutionary model were favored, we initially

adjusted the branch lengths such that the sum of

branch lengths was equal between the compared

topologies and that the root was approximately at the

same age (in this case in the Tournaisian, the first

stage of the Carboniferous). This was done for the

trees used to compare the hypotheses using the

cranial dataset because if a model incorporating

(variable) branch length information had been select-

ed, and if the trees representing the various hypothe-

ses had not all had the same total length (the sum of

all branch lengths), the resulting distortions in branch

lengths created around the extinct taxa (whose height

compared to extant taxa is specified by their geolo-

gical age) would have introduced another variable

influencing the AICc. But given that the selected

model ignores branch lengths, this precaution turned

out to be superfluous. We have therefore not made

these time-consuming adjustments to the additional

trees we generated later to analyze the appendicular

data.

RESULTS

In the phylogenetic analysis of cranial data, a

single tree island of 22,077 trees of 438 steps was

found, only once, so there might be more trees of that

length and perhaps even shorter trees. Initially, an is-

land of 22,075 trees was found; we swapped on each

of these in a subsequent run, which only recovered

two additional trees. Given that slightly longer trees

did not differ much from those that we obtained, the

low quality of the results (poor congruence with the

established consensus about the monophyly of major

clades such as squamates, birds, mammals and

turtles) and the fact that about four full days of

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computer time had been spent on analysis of the

cranial data, we did not pursue that search further. As

expected, the strict consensus tree is poorly resolved

(Fig. 3). The majority-rule consensus (not shown, but

available in the supplements available on the bioRχiv

web page) is more resolved but not necessarily better

because much of the additional resolution contradicts

the established consensus. For the appendicular

matrix, 22,757 trees of 164 steps were found. Their

strict consensus (Fig. 4) deviates even more from the

established consensus than the tree obtained from

cranial data.

Figure 3. Strict consensus of the most parsimonious trees obtained by analyzing cranial dataset 2, which is comprised of

105 taxa and seven characters (see Table 1). Note that several higher taxa whose monophyly is well-established are para-

or polyphyletic here.

This visual assessment of phylogenetic signal

through an examination of the consensus trees (Figs.

3, 4) is congruent with the test based on squared-

change parsimony and random taxon reshuffling

(Laurin 2004). Indeed, the latter indicates that the

phylogenetic signal in the cranial data is fairly strong,

with a probability of less than 0.0001 that the ob-

served covariation between the data and the tree re-

flects a random distribution (none of the 10,000 ran-

dom trees generated were as short as the reference

tree). However, it is weaker, with a probability of

0.0017, for the appendicular data.

The speciational model of evolution, in which all

branch lengths are equal, has overwhelming support

among cranial data, whether or not the Permian tem-

nospondyl Sclerocephalus (Table 2) or the squamo-

sal (Table 3) are included (including Sclerocephalus

adds a second temnospondyl genus, but given that

the timing of ossification of the squamosal is unknown

in Sclerocephalus, including it requires excluding the

squamosal from the analysis as described in the

Methods section); the five other examined models

have AICc weights < 10-11. For the appendicular data,

the speciational model also has the most support, but

that support is not as strong and varies depending on

which dataset is analyzed (seven characters or four)

and under which phylogenetic hypothesis. In three of

the four tests performed, support for the second-best

model, the non-phylogenetic/equal model, varied

between 5% and 19% (Table 4).

Two main conclusions can be drawn from these

tests (Tables 2–4). First, given that both of the best-

supported models imply equal branch lengths, actual

time represented by branches can be ignored, so we

compare support of the six competing topologies

using only the best-supported model (speciational).

This simplifies the discussion, because it means that

the original branch lengths are irrelevant (under that

model, all branch lengths are equal); unfortunately,

the branch length (evolutionary time) data were need-

ed to reach this conclusion. Thus, the only remaining

variable is the topology. Second, model fitting, along

with the test based on squared-change parsimony

and random taxon reshuffling, indicates that the

phylogenetic signal in the cranial data is strong, but

that it is noticeably weaker in the appendicular data

(this is shown mostly by the non-negligible support for

the non-phylogenetic/equal model). Thus, compari-

sons of the fit of the various phylogenetic hypotheses

for the cranial data should be more reliable than for

the appendicular data. However, given that for sev-

eral Paleozoic taxa (most importantly both of the sam-

pled lepospondyls), comparisons can be performed

only for the appendicular data, these were performed

as well.

Strict, cranial analysis 6, 438 steps

Apateon pedestris (Erdesbach)

Apateon caducus (Erdesbach)

Monodelphis domestica*

Zootoca vivipara*

Caluromys philander*

Perameles nasuta*

Rhabdomys pumilio*

Mesocricetus auratus*

Cavia porcellus*

Tupaia javanica*

Felis catus*

Mogera wogura*

Cryptotis parva*

Erinaceus amurensis*

Pseudacris triseriata

Triprion petasatus

Rattus norvegicus (albino)*

Loxodonta africana*

Didelphis albiventris*

Macropus (Notomacropus) eugenii*

Dasyurus viverrinus*

Peromyscus melanophrys*

Alligator mississipiensis*

Trichosurus vulpecula*

Meriones unguiculatus*

Gegeneophis ramaswamii

Andrias japonicus

Salamandra salamandra

Salamandrella keyserlingii

Ambystoma tigrinum

Eleutherodactylus coqui

Pleurodeles waltl

Lissotriton vulgaris*

Hypogeophis rostratus

Eleutherodactylus nubicola*

Pelodiscus sinensis*

Apalone spinifera*

Ambystoma mexicanum

Lerista bougainvilii*

Somateria mollissima

D. novaehollandiae (YPM)

Hemiergis peronii*

Coturnix coturnix (N&T)*

Dromaius novaehollandiae (RM)*

Gallus gallus (Maxwell)

Meleagris gallopavo*

Struthio camelus*

Cairina moschata

Sterna hirundo

Rousettus amplexicaudatus*

Chelydra serpentina*

Liopholis whitii*

Saiphos equalis*

Homo sapiens*

Stercorarius skua*

Larus ridibundus

L. canus

Phalacrocorax auritus*

C. coturnix (Maxwell)*

L. argentatus

Echinops telfairi*

Pipa myersi

Tamandua tetardactyla*

Procavia capensis*

Heterohyrax brucei*

Tenrec ecaudatus*

Eremitalpa granti*

Orycteropus afer*

Talpa spp. (to remove)*

Bradypus variegatus*

Dasypus novemcinctus*

Anas platyrhynchos

Mus musculus*

Uperoleia laevigata

Dicamptodon tenebrosus

Rhyacotriton cascadeum

Eurycea bislineata

Siren intermedia

Notophthalmus viridescens

Ambystoma maculatum

Ambystoma talpoideum

Onychodactylus japonicus

Ranodon sibiricus

Gyrinophilus porphyriticus

Hemidactylium scutatum

Amphiuma means

Pseudophryne bibroni

Crinia signifera

Anaxyrus boreas

Rana (Amerana) cascadae

Hyla versicolor

Philautus silus*

Spea bombifrons

Smilisca baudini

Osteopilus septentrionalis

Rana (Rana) temporaria

Rana (Amerana) pretiosa

Pseudis platensis

Rhinophrynus dorsalis*

Alytes obstetricans*

Hymenochirus boettgeri

Hypsiboas lanciformis

Ascaphus truei*

Pseudacris regilla

Rana (Amerana) aurora

Character 1: Taxa

Parsimony

reconstruction (

Unordered) [Steps: 23]

actinopterygians

temnospondyls

lepospondyls

gymnophionans

urodeles

anurans

turtles

squamates

archosaurs

mammals

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The copyright holder for this preprint. http://dx.doi.org/10.1101/352609doi: bioRxiv preprint first posted online Jun. 20, 2018;

Preprint uploaded to bioRχiv on 08 October 2019

Figure 4. Strict consensus of the most parsimonious trees obtained by analyzing appendicular dataset 3, which is comprised

of 62 taxa and seven characters (see Table 1). The phylogenetic signal in these data seems to be lower than in the cranial

data.

Using the speciational model, the AICc weights of

the six compared topologies indicate that there is

strong support in the cranial data for the LH (lepo-

spondyl hypothesis), with an AICc weight of 0.9885

when Sclerocephalus is included (Table 5) and

0.8848 when the squamosal is included instead

(Table 6). Of the other topologies, the TH (temno-

spondyl hypothesis) was by far the best supported,

with an AICc weight of 0.01144 (with Sclerocephalus)

or 0.1056 (with the squamosal), which is 86.44 or

8.38 times less than for the LH. Both versions of the

DH (diphyly hypothesis) and of the PH (polyphyly

hypothesis) have negligible support (AICc weights <

0.01 when the squamosal is included, < 0.0001 when

Sclerocephalus is included). The least support is

found for the PH2 when Sclerocephalus is included,

and for the DH1 when the squamosal is included. In

both cases, the recently proposed DH2 (Pardo et al.

2017b) fares second-worst by a small margin.

Notably, the DH1 contradicts the modern consensus

on lissamphibian monophyly (Fig. 1g), while the PH2

and the DH2 fulfill this constraint from the molecular

but not the paleontological point of view, having

lissamphibian monophyly with respect to amniotes

but not with respect to temnospondyls (Fig. 1f, h).

A slightly different dataset is used (only 84 taxa,

but eight cranial characters – excluding the squamo-

sal but including the frontal and the vomer – and

Apateon sequences for both species from Erdesbach

rather than Obermoschel) provides even stronger

support for the LH, with an AICc weight of 0.9935

(Table 7). The next best-supported topology, which

simultaneously represents the TH, DH1 and DH2

(due to the absence of caecilians from this dataset),

has an AICc weight of only 0.0065.

Strict, postcranial analysis

Eusthenopteron foordi

Pipa pipa

Apateon pedestris (Obermoschel)

Apateon caducus (Obermoschel)

Hylorina sylvatica*

Didelphis albiventris*

Saiphos equalis*

Pelobates cultripes*

Hypsiboas

Fejervarya cancrivora*

Chelydra serpentina*

Epipedobates tricolor*

Cornufer guentheri*

Xenopus laevis

Liopholis whitii*

Palaeobatrachus

Dendrobates auratus

Spea multiplicata

Hamptophryne boliviana*

Discoglossus sardus

Phyllomedusa vaillanti

Lerista bougainvilii*

Ambystoma mexicanum

Kassina senegalensis*

Bufo bufo

Sclerocephalus haeuseri

Archegosaurus decheni

Micromelerpeton credneri

Hyloplesion longicostatum*

Microbrachis pelikani

Ambystoma macrodactylum

Bombina orientalis

Scaphiopus

Spea bombifrons

Epidalea calamita*

Ceratophrys cornuta

Eleutherodactylus nubicola*

Rana (Pantherana) pipiens

Leptodactylus chaquensis*

Chacophrys pierotti*

Echinops telfairi*

Tenrec ecaudatus*

Meleagris gallopavo*

Coturnix coturnix (N&T)*

C. coturnix (Maxwell)*

Struthio camelus*

D. novaehollandiae (YPM)

Anas platyrhynchos

Cairina moschata

Somateria mollissima

Larus ridibundus

L. canus

Phalacrocorax auritus*

Gallus gallus (Maxwell)

Gallus gallus (S&W)

Sterna hirundo

Stercorarius skua*

Hemiergis peronii*

Pyxicephalus adspersus*

Dromaius novaehollandiae (RM)*

L. argentatus

Alligator mississipiensis*

Character 1: Taxa

Parsimony

reconstruction (

Unordered) [Steps: 28]

actinopterygians

temnospondyls

lepospondyls

gymnophionans

urodeles

anurans

turtles

squamates

archosaurs

mammals

(which was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.

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Table 2. Support (AICc and AICc weights) for six evolutionary models given our reference tree (LH) and dataset 1 (see Table

1), which comprises six cranial characters (nasal, parietal, squamosal, maxilla, pterygoid, and exoccipital) scored in 107

taxa, including the temnospondyl Sclerocephalus. This was performed on the tree representing the LH (lepospondyl

hypothesis), but doing this on other trees leads to similar results. Numbers presented with four significant digits; best values

in boldface. “Distance” refers to keeping the original branch lengths (which represent evolutionary time), “equal” sets all

branch lengths (internal and terminal) to 1, “free” infers them from the data. Abbreviations: k, number of estimable para-

meters; l, likelihood; wi, weight; ∆i, difference of AICc from that of the Pure-Phylogenetic / Equal model.

Evolutionary model

AIC

AICc

∆i AICc

wi(AICc)

Pure-Phylogenetic / Distance

−584.4

293.2

−583.4

641.2

5.85 E−140

Pure-Phylogenetic / Equal

(speciational)

−1225.6

613.8

−1224.6

1.000

Pure-Phylogenetic / Free

2.000 E10

−1.000 E10

486

2.000 E10

< E−165

Non-Phylogenetic / Distance

−473.6

237.8

−472.6

752.0

4.97 E−164

Non-Phylogenetic / Equal

−959.9

481.0

−958.9

265.7

2.02 E−58

Non-Phylogenetic / Free

2.000 E10

−1.000 E10

244

2.000 E10

< E−165

Table 3. Support (AICc and AICc weights) for six evolutionary models given our reference tree (LH) and dataset 2 (see Table

1), which comprises seven cranial characters (nasal, parietal, squamosal, premaxilla, maxilla, pterygoid, and exoccipital)

and 105 taxa, excluding Sclerocephalus. Abbreviations and boldface as in Table 2.

Evolutionary model

AIC

AICc

∆i AICc

wi(AICc)

Pure-Phylogenetic / Distance

−715.9

359.0

−714.9

683.5

< E−26

Pure-Phylogenetic / Equal

−1399.5

700.7

−1398.5

1.000

Pure-Phylogenetic / Free

2.000 E10

−1.000 E10

306

2.000 E10

Non-Phylogenetic / Distance

−580.6

291.3

−579.6

818.8

< E−26

Non-Phylogenetic / Equal

−1106.0

554.0

−1105.0

293.5

2.278 E−98

Non-Phylogenetic / Free

2.000 E10

−1.000 E10

244

2.000 E10

< E−26

Table 4. AICc weights showing relative support for six evolutionary models given various appendicular datasets (3 and

4; see Table 1) and various hypotheses. Because of the number of analyses presented below, only the AICc weights are

presented (best values in boldface). Abbreviations: DH, diphyly hypothesis (both versions); LH, lepospondyl hypothesis; TH,

temnospondyl hypothesis.

Evolutionary model

7 characters, LH

4 characters, LH

4 characters, TH/DH

Pure-Phylogenetic / Distance

5.1857 E−149

2.340 E−70

1.227 E−52

2.646 E−52

Pure-Phylogenetic / Equal

0.9335

0.94459

0.8139

Pure-Phylogenetic / Free

< E−179

1.598 E−277

4.012 E−158

3.002 E−155

Non-Phylogenetic / Distance

7.515 E−179

4.843 E−52

2.162 E-42

7.262 E−42

Non-Phylogenetic / Equal

2.14914 E−64

6.648 E−02

5.541 E−02

0.1861

Non-Phylogenetic / Free

< E−179

The appendicular data are available in far more

Paleozoic taxa than the cranial data; these include

Sclerocephalus haeuseri, Archegosaurus decheni,

and the non-branchiosaurid “branchiosaur” Micromel-

erpeton credneri among temnospondyls, the lepo-

spondyls Hyloplesion longicaudatum and Microbra-

chis pelikani, and the tristichopterid finned stem-tetra-

podomorph Eusthenopteron foordi, in addition to the

same two species of Apateon as for the cranial data-

sets, A. caducus and A. pedestris. Analysis of these

data (seven characters: humerus, radius, ulna, ilium,

femur, tibia and fibula) yields surprising results, with

the PH2 having the most support, with an AICc weight

of 0.7978 when using the dataset of seven bones

(Table 8). The TH, DH1 and DH2 with “branchiosaur”

monophyly are collectively (they cannot be distin-

guished with that taxonomic sample) the second-best

hypotheses with that dataset, with an AICc weight of

only 0.1874. The least-supported hypothesis with

these data is the TH with “branchiosaur” polyphyly.

Using the other postcranial dataset with only four

bones (radius, ulna, ilium, and femur) but with more

taxa (notably the branchiosaurid temnospondyl

“Melanerpeton” humbergense) shows that intraspeci-

fic variation in the postcranial ossification sequences

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of Apateon do not significantly impact our assess-

ment of the support for various hypotheses. Whether

both sequences of Apateon (from the Erdesbach and

Obermoschel localities, which represent separate

paleo-lakes) are included (treated as if they were

distinct taxa, such as subspecies), or whether either

one of these is used in isolation, the PH2 retains the

highest support, with AICc weights of 0.62 to 0.65.

The LH is a distant second, at 0.20–0.23, but still well

ahead of the TH/DH and the PH1, which all receive

AICc weights between 0.03 and 0.06 (Table 9).

Table 5. Support (AIC and AICc weights) for the six topologies, reflecting the six hypotheses about the origin of extant

amphibians, under the speciational model (called Pure-Phylogenetic / Equal in Tables 2–4), with dataset 1 (see Table 1),

which includes six cranial characters (nasal, parietal, squamosal, maxilla, pterygoid, and exoccipital) and 107 taxa (including,

among Paleozoic taxa, Apateon and Sclerocephalus). Abbreviations and boldface as in Table 2, except ∆i: difference of

AICc from that of the LH. Hypotheses from top to bottom: LH: monophyletic origin from lepospondyls; TH: monophyletic

origin among temnospondyls; DH1: diphyletic origin, caecilians from lepospondyls and batrachians from temnospondyls, as

in Anderson et al. (2008); DH2: diphyletic origin (batrachians and caecilians from different temnospondyls: Pardo et al.

2017b); PH1: triphyletic (polyphyletic) origin with anurans and urodeles from different temnospondyls, caecilians from lepo-

spondyls, and lepospondyls closer to Amniota than to Batrachia (Fröbisch et al. 2007); PH2: triphyletic (polyphyletic) origin

as above, but with lepospondyls and caecilians closer to temnospondyls than to amniotes (Milner 1993), reflecting the well-

established lissamphibian monophyly among extant taxa (e.g. Irisarri et al. 2017; Feng et al. 2017).

Hypothesis

AIC

AICc

∆i AICc

wi(AICc)

−1217

609.4

−1215

8.919

0.01144

−1226

613.8

−1224

0.9885

DH1

−1204

602.9

−1202

21.90

1.738 E−05

DH2

−1195

598.3

−1193

31.01

1.827 E−07

PH1

−1194

597.9

−1192

31.86

1.196 E−07

PH2

−1193

597.4

−1191

32.89

7.143 E−08

Table 6. Support (AIC and AICc weights) for the six topologies, reflecting the six hypotheses about the origin of extant

amphibians, for dataset 2 (see Table 1), which includes seven cranial characters (nasal, parietal, squamosal, premaxilla,

maxilla, pterygoid, and exoccipital) and 105 taxa, excluding Sclerocephalus (among Paleozoic taxa, only Apateon is present).

Abbreviations, boldface and hypotheses as in Tables 2 and 5.

Hypothesis

AIC

AICc

∆i AICc

wi(AICc)

−1395

698.6

−1394

4.251

0.1056

−1399

700.7

−1398

0.8848

DH1

−1384

693.1

−1383

15.203

4.42 E−4

DH2

−1385

693.6

−1384

14.315

6.89 E−4

PH1

−1387

694.5

−1386

12.404

1.792 E−3

PH2

−1390

695.8

−1388

9.792

6.615 E−3

Table 7. Support for the various hypotheses about amphibian origins for dataset 5 (see Table 1), which includes eight cranial

characters (frontal added) and 84 taxa, with Apateon sequences from Erdesbach (in addition to Sclerocephalus among

Paleozoic taxa). Abbreviations, boldface and hypotheses as in Tables 2 and 5. Because of the taxon sample, only three

topologies can be tested.

Hypothesis

AIC

AICc

∆i AICc

wi(AICc)

−1296

649.0

−1294

0.9935

TH, DH1, DH2

−1286

644.0

−1284

10.061

6.493 E−3

−1274

638.0

−1272

22.038

1.628 E−5

DISCUSSION

Phylogenetic signal

In his discussion of previous phylogenetic conclu-

sions from ossification sequences (e.g. Schoch and

Carroll 2003), Anderson (2007) noted that ossification

sequences seemed to abound in symplesiomorphies

and in autapomorphies of terminal taxa, while poten-

tial synapomorphies were scarce. This pessimism

was seemingly confirmed by Schoch (2006) in a

paper that was published after Anderson’s (2007)

book chapter had gone to press: not only were many

similarities in the cranial ossification sequences

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across Osteichthyes found to be symplesiomorphies,

but a phylogenetic analysis of cranial ossification

sequences did not recover Mammalia, Sauropsida,

Amniota or Lissamphibia as monophyletic. Along with

these results, Schoch (2006) dismissed another: the

position of the temnospondyl Apateon caducus (the

only included extinct taxon) outside the tetrapod

crown-group, i.e. the lepospondyl hypothesis on liss-

amphibian origins (LH).

While ossification sequences alone may not pro-

vide enough data for a phylogenetic analysis, as

shown by our results (Fig. 3, 4), there is clearly a phy-

logenetic signal because the taxa are not randomly

scattered over the tree. Specifically, our datasets

(with much larger taxon samples than in Schoch

2006) fit some tree topologies much better than

others. Both the tests using CoMET and squared-

change parsimony with random taxon reshuffling

overwhelmingly support the presence of a strong

phylogenetic signal in the cranial data; the null

hypothesis of the absence of a phylogenetic signal

can be rejected in both cases, given that it has a

probability of < 10-97 for the cranial and < 10-4 for the

appendicular dataset. We conclude that the cranial

dataset contains a strong phylogenetic signal, and

are therefore cautiously optimistic about future contri-

butions of ossification sequences to phylogenetics.

We are less optimistic about the appendicular se-

quence data, which both tests suggest contains less

phylogenetic signal.

The sizable effect on nodal estimates and inferred

heterochronies of intraspecific variation found by

Sheil et al. (2014) in lissamphibians could raise

doubts about the robustness of our findings. We have

been able to incorporate infraspecific variability in

only two terminal taxa (Apateon caducus and A.

pedestris), but Apateon has played a prominent role

in discussions about the significance of cranial

ossification sequences on the origins of extant

amphibians (Schoch and Carroll 2003; Schoch 2006;

Germain and Laurin 2009). Thus, incorporation of

intraspecific variability in Apateon is presumably

much more important than in extant taxa, even

though variability in the latter would obviously add to

the analysis and should be tackled in the future. The

variability in Apateon should be exempt from two

sources of artefactual variability in ossification se-

quences discussed by Sheil et al. (2014), namely the

way in which the specimens were collected (there can

be no lab-raised specimens in long-extinct taxa) and

the fixing method used (in this case, fossilization

under quite consistent taphonomic conditions). The

finding that the results are very similar no matter

whether we used the Apateon sequences from Erdes-

bach, Obermoschel, or both, we find very similar re-

sults (Table 9), is reassuring. In this case,

intraspecific variation has negligible impact. How-

ever, future studies should attempt to assess the ef-

fect of more generalized incorporation of infraspecific

variability (in a greater proportion of the OTUs).

Of course, these results do not preclude functional

or developmental constraints from applying to the

same data. This phenomenon has been documented,

among other taxa, in urodeles, whose development

has often been compared with that of temnospondyls

(e.g. Schoch 2006; Schoch and Carroll 2003; Frö-

bisch et al. 2007, 2015; Germain and Laurin 2009).

For instance, Vorobyeva and Hinchliffe (1996) docu-

mented the larval functional constraints linked to early

forelimb use that may cause an early development of

manual digits 1 and 2, compared with other tetrapods,

as briefly discussed below. However, in the case of

our seven cranial characters, there is no evidence of

functional constraints. This is a little-investigated

topic, but all these bones apparently form a single

developmental module of the urodele skull (Laurin

2014). For the appendicular data, functional con-

straints might explain the more subdued phylogenetic

signal, but this will have to be determined by

additional research.

The finding that the postcranial characters that we

analyzed contain relatively little phylogenetic signal

may raise doubts about the claims that have been

made about the phylogenetic implications of other

such data. Specifically, Carroll et al. (1999) stated

that the neural arches ossify before the centra in frogs

and temnospondyls, but not in salamanders, caecili-

ans or lepospondyls. When it was found that the cen-

tra do ossify first in a few cryptobranchoid salaman-

ders, Carroll (2007: 30) took this as “strong evidence

that the most primitive crown-group salamanders had

a sequence of vertebral development that is common

to frogs and labyrinthodonts [including temnospon-

dyls] (but distinct from that of lepospondyls)”. In fact,

apart from tail regeneration in Hyloplesion and Micro-

brachis (where the centra ossify before the neural

arches: Olori 2015; Fröbisch et al. 2015; van der Vos

et al. 2017), only one incompletely ossified vertebral

column (referred to Utaherpeton) is known of any pu-

tative lepospondyl. “In this specimen, […] five neural

arches […] have ossified behind the most posterior

centrum.” (Carroll and Chorn 1995: 40–41) Carroll’s

(2007: 85) claim that “the centra always ossified prior

to the arches” in lepospondyls is therefore rather

puzzling.

Fröbisch et al. (2007, 2015) pointed out that the

first two digital rays (digits, metapodials and distal

carpals/tarsals) ossify before the others (“preaxial

polarity”) in salamanders and the temnospondyls

Apateon, Micromelerpeton and Sclerocephalus, while

(which was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.

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Laurin et al. 2019 — Lissam phibian origin and ossification sequences

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the fourth ossifies first (“postaxial polarity”) in amni-

otes, frogs and “probably” (Fröbisch et al. 2015: 233,

234) the lepospondyls Microbrachis and Hyloplesion.

This latter inference, however, is based only on a

delay in the ossification of the fifth ray that is shared

specifically with sauropsid amniotes (Olori 2015).

Ossification sequences (however partial) of the other

four rays in any lepospondyl are currently limited to

the tarsus of Batropetes, which clearly shows preaxial

polarity (Glienke 2015: fig. 6O–S; Marjanović and

Laurin 2019), and that of the putative (but see Clack

et al. 2019) lepospondyl Sauropleura, in which like-

wise the second distal tarsal ossified before all others

(Marjanović and Laurin 2019). Outside of temno- and

lepospondyls, Marjanović and Laurin (2013, 2019)

presented evidence that preaxial polarity is plesio-

morphic, widespread and dependent on the use of the

still developing limbs for locomotion, which would

explain why it was independently lost in amniotes and

frogs and reduced (the second ray still forms first, but

the delays between the rays are much reduced so

that all form nearly at the same time) in direct-devel-

oping salamanders as well as in th elimb regeneration

of terrestrial postmetamorphic salamanders (Kumar

et al. 2015). It may be relevant here that the PH2 (Fig.

1f), favored by our appendicular data, groups exactly

those sampled taxa in a clade that are known to have

preaxial polarity in limb development. To sum up,

neither our own analyses nor the previous works that

we cited above demonstrated conclusively that ossifi-

cation sequences of postcranial elements provide

reliable clues about the origin of extant amphibians.

Table 8. Support (AICc weights) for the various hypotheses about amphibian origins according to dataset 3 (see Table 1),

which features seven appendicular characters (humerus, radius, ulna, ilium, femur, tibia and fibula) and 62 taxa, including

several Paleozoic taxa (the temnospondyls Archegosaurus decheni and Micromelerpeton credneri, the lepospondyls Hylo-

plesion longicaudatum and Microbrachis pelikani, and the tristichopterid Eusthenopteron foordi) in addition to Apateon (two

species, A. caducus and A. pedestris) and Sclerocephalus haeuseri. The Apateon sequences come from Obermoschel.

Abbreviations, boldface and hypotheses as in Table 5, except that the TH and both variants of the DH become indistinguish-

able, but the phylogenetic position of the “branchiosaur” Micromelerpeton can be tested.

Hypothesis

AIC

AICc

∆i AICc

wi(AICc)

−885.0

443.5

−884.2

11.808

2.177 E−3

TH, DH (branchiosaur monophyly)

−881.1

441.6

−880.3

2.897

0.1874

TH, DH (branchiosaur polyphyly)

−886.4

444.2

−885.6

15.754

3.027 E−4

PH1

−888.5

445.3

−887.7

8.341

0.01232

PH2

−896.9

449.4

−896.1

0.000

0.7978

Table 9. Effect of the intraspecific variability in ossification sequences of Apateon on the support (AICc weight; best values

in boldface) for the various hypotheses about amphibian origins. The dataset (number 4; Table 1) includes only four appendi-

cular bones (radius, ulna, ilium, and femur) and 63 to 65 taxa but it allows testing the impact of intraspecific variability in

ossification sequences in Apateon, which are documented in two localities (Erdesbach and Obermoschel). Because of the

number of tests presented (15: five topologies x three sets of sequences), only the AICc weights are given. In all tests, the

following Paleozoic taxa are present: Sclerocephalus haeuseri, Archegosaurus decheni, “Melanerpeton” humbergense,

Micromelerpeton credneri, Apateon (two species, A. caducus and A. pedestris) among temnospondyls, Hyloplesion longicau-

datum and Microbrachis pelikani among lepospondyls, and the tristichopterid Eusthenopteron foordi. For abbreviations of

the hypotheses, see Table 5.

Hypothesis

Erdesbach and

Obermoschel

Erdesbach

Obermoschel

0.21407

0.20169

0.22657

TH, DH (branchiosaur monophyly)

0.05492

0.05265

0.05532

TH, DH (branchiosaur polyphyly)

0.03713

0.04285

0.03342

PH1

0.05653

0.05491

0.05638

PH2

0.63735

0.64790

0.62832

In contrast, we are reasonably confident about our

results on the cranial ossification sequences. Given

the phylogenetic signal we have found in our cranial

datasets, we think that ossification sequence data

should eventually be added to phenotypic datasets

for analyses of tetrapod phylogeny. Indeed, an analy-

sis of amniote phylogeny using data from organoge-

nesis sequences (coded using event-pairing in Parsi-

mov) already exists (Werneburg and Sánchez-Villa-

(which was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint. http://dx.doi.org/10.1101/352609doi: bioRxiv preprint first posted online Jun. 20, 2018;

Laurin et al. 2019 — Lissam phibian origin and ossification sequences

PREPRINT

gra 2009). The usefulness of such data for phyloge-

netic inference was further tested, with encouraging

results, by Laurin and Germain (2011), and the

present analysis adds additional support for it.

Indirect support for the lepospondyl

hypothesis from temnospondyls

The strong support for the lepospondyl hypothesis

that we have found in cranial data is surprising be-

cause cranial ossification sequence data, especially

those of the Permo-Carboniferous temnospondyl

Apateon, have often been claimed to contradict the

LH (lepospondyl hypothesis, Fig. 1d). Similarities be-

tween Apateon and extant urodeles, in particular the

supposedly “primitive” hynobiid Ranodon, have often

been emphasized (Schoch and Carroll 2003; Schoch

and Milner 2004; Carroll 2007; Schoch 2014b). How-

ever, other studies have already raised doubts about

some of these claims (e.g. Schoch 2006; Anderson

2007; Germain and Laurin 2009). Schoch (2006) and

Anderson (2007) concluded that most characters

shared between Apateon and urodeles were plesio-

morphies. Germain and Laurin (2009) also demon-

strated that, far from being very similar to the ances-

tral urodele morphotype (contra Schoch and Carroll

2003 or Carroll 2007), the cranial ossification se-

quence of Apateon was statistically significantly dif-

ferent from that of the hypothetical last common

ancestor of all urodeles (as suspected by Anderson

2007). However, these earlier studies did not clearly

show which of the various hypotheses on lissamphi-

bian origins the ossification sequences of Apateon

spp. – or the newly available partial sequence (Wer-

neburg 2018) of the phylogenetically distant temno-

spondyl Sclerocephalus – supported most. This is

what we have attempted to do here.

Unfortunately, the development of lepospondyls is

too poorly documented to be incorporated into the

cranial analyses, but we included two lepospondyls in

analyses of appendicular data. These analyses

weakly favor a polyphyletic origin of extant amphibi-

ans, with both temno- and lepospondyls in the amphi-

bian clade, a hypothesis that has not been advocated

seriously for decades (Milner 1993: fig. 5B) as far as

we know. However, given the moderate phylogenetic

signal in these data, we view these results with

skepticism. Olori (2011), using event-pairing with Par-

simov (Jeffery et al. 2005) and PGi (Harrison and

Larsson 2008), analyzed lepospondyl postcranial

ossification sequences and concluded that support

for the three hypotheses that she tested (TH/DH with

two different positions for Micromelerpeton, and LH)

did not differ significantly. By contrast, our analyses

of the postcranial data indicate a stronger support for

polyphyly (PH2) than for the TH/DH, which is only a

distant second (Table 8) or third (behind PH2 and LH;

Table 9) depending on the analyses. Olori (2011)

performed no statistical test of phylogenetic signal of

her data, though a related test (performing phyloge-

netic analyses on the data) yielded trees (Olori, 2011:

fig. 5.5–5.7) that are largely incongruent with the

established consensus, in which most large taxa

(Mammalia, Testudines, Lissamphibia, etc.) are para-

or polyphyletic. Olori’s (2011) results, like ours, sup-

port the conclusion that the phylogenetic signal in

postcranial ossification sequence data is low.

Given the current limitations in the availability of

developmental data in Paleozoic stegocephalians,

we hope to have demonstrated that cranial ossifica-

tion sequences of amniotes, lissamphibians and

temnospondyls provide support for the LH that is

independent of the phylogenetic analyses of Laurin

(1998), Pawley (2006: appendix 16) or Marjanović

and Laurin (2009, 2019). This independence is impor-

tant because the cranial ossification sequence data

cannot rival the morphological data in terms of data

availability, simply because growth sequences of

extinct taxa are rare (Sánchez-Villagra 2012), but

having a fairly independent line of evidence to investi-

gate a major evolutionary problem is clearly advanta-

geous. We hope that the modest methodological

progress made in this study will stimulate the search

for fossilized ontogenies (Cloutier 2009; Sánchez-

Villagra 2010).

ACKNOWLEDGEMENTS

Jennifer Olori, two anonymous reviewers and the

editor Robert Asher made helpful comments that

improved this paper. D. M. would further like to thank

Ralf Werneburg for an electronic reprint of his 2018

paper, Nadia Fröbisch for discussion of limb develop-

ment in salamanders, and Daniel Field for discussion

of molecular divergence times and the fossil record.

ADDITIONAL INFORMATION

Funding

This work was supported by the Centre National

de la Recherche Scientifique and the French Ministry

of Research (unnumbered recurring grants to the

CR2P, for ML).

Competing interests

None that we are aware of.

Author contributions

ML designed the study, supervised the data

collection, analyzed the data and wrote much of the

draft; OL collected most of the ossification sequence

(which was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint. http://dx.doi.org/10.1101/352609doi: bioRxiv preprint first posted online Jun. 20, 2018;

Laurin et al. 2019 — Lissam phibian origin and ossification sequences

PREPRINT

data; DM added data to our database (mostly of

Paleozoic taxa), updated the timetrees, participated

in the writing and drafted Figure 1.

Data availability

All data used in this study can be downloaded from

the same bioRχiv page as this preprint.

Supplementary information

Data matrices and trees used in this analysis can

be downloaded in NEXUS format for Mesquite from:

https://www.biorxiv.org/content/10.1101/352609v3.s

upplementary-material

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Appendix 1: Sources of data for ossification sequences.

Empty cells indicate that these data are unavailable. Three methods were examined, and we used the one for

which most data were available (position in the ossification sequence, last column).

Standardization method (data type used)

Taxa

Ontogenetic stages

Snout-vent length

(mm)

Ossification sequence

position

Actinopterygii

Amia calva

Grande and Bemis

1998

Grande and Bemis 1998

Clarias gariepinus

Adriaens and Verraes

1998

Adriaens and Verraes 1998

Danio rerio

Cubbage and Mabee

1996

Cubbage and Mabee 1996

Oryzias latipes

Langille and Hall 1987

Tristichopteridae

Eusthenopteron foordi

Cote et al. 2002;

Leblanc and Cloutier

2005

Cote et al. 2002; Leblanc

and Cloutier 2005

Temnospondyli

Archegosaurus decheni

Witzmann 2006

Apateon caducus (Erdesbach)

Schoch 2004

Apateon caducus (Obermoschel)

Werneburg 2018

Apateon pedestris (Erdesbach)

Schoch 2004

Apateon pedestris (Obermoschel)

Werneburg 2018

“Melanerpeton” humbergense

Schoch 2004

Micromelerpeton credneri

Boy 1995; Lillich and

Schoch 2007;

Witzmann and

Pfretzschner 2009;

Schoch 2009

Boy 1995; Lillich and

Schoch 2007; Witzmann

and Pfretzschner 2009;

Schoch 2009

Sclerocephalus haeuseri

Lohmann and Sachs

2001; Schoch 2003;

Schoch and Witzmann

2009; Werneburg 2018

Lohmann and Sachs

2001; Schoch 2003;

Schoch and Witzmann

2009; Werneburg 2018

Lohmann and Sachs 2001;

Schoch 2003; Schoch and

Witzmann 2009; Werneburg

2018

Lepospondyli

Hyloplesion longicaudatum

Olori 2013

Microbrachis pelikani

Olori 2013

Gymnophiona

Gegeneophis ramaswamii

Müller et al. 2005

Harrington et al. 2013

Hypogeophis rostratus

Müller 2006

Harrington et al. 2013

Urodela

Aneides lugubris

Wake et al. 1983

Ambystoma macrodactylum

Harrington et al. 2013

Ambystoma maculatum

Moore 1989

Harrington et al. 2013

Ambystoma mexicanum

Laurin and Germain

2011

Harrington et al. 2013

Ambystoma talpoideum

Reilly 1987

Ambystoma texanum

Laurin and Germain

2011

Harrington et al. 2013

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Taxa

Standardization method (data type used)

Ontogenetic stages

Snout-vent length

(mm)

Ossification sequence

position

Ambystoma tigrinum

Harrington et al. 2013

Amphiuma means

Harrington et al. 2013

Andrias japonicus

Harrington et al. 2013

Bolitoglossa subpalmata

Ehmcke and Clemen 2000

Dicamptodon tenebrosus

Harrington et al. 2013

Eurycea bislineata

Harrington et al. 2013

Gyrinophilus porphyriticus

Harrington et al. 2013

Hemidactylium scutatum

Harrington et al. 2013

Lissotriton vulgaris

Laurin and Germain

2011

Harrington et al. 2013

Necturus maculosus

Harrington et al. 2013

Notophthalmus viridescens

Reilly 1986

Harrington et al. 2013

Onychodactylus japonicus

Harrington et al. 2013

Pleurodeles waltl

Harrington et al. 2013

Ranodon sibiricus

Harrington et al. 2013

Salamandra salamandra

Harrington et al. 2013

Salamandrella keyserlingii

Harrington et al. 2013

Siren intermedia

Reilly and Altig 1996

Triturus karelinii

Harrington et al. 2013

Anura

Alytes obstetricans

Yeh 2002

Ascaphus truei

Harrington et al. 2013

Anaxyrus boreas

Gaudin 1978

Bombina orientalis

Harrington et al. 2013

Bufo bufo

Harrington et al. 2013

Cornufer guentheri

Harrington et al. 2013

Ceratophrys cornuta

Harrington et al. 2013

Chacophrys pierotti

Harrington et al. 2013

Crinia signifera

Harrington et al. 2013

Dendrobates auratus

de Sá and Hill 1998

Harrington et al. 2013

Discoglossus sardus

Pugener and Maglia 1997

Eleutherodactylus coqui

Harrington et al. 2013

Eleutherodactylus nubicola

Harrington et al. 2013

Epidalea calamita

Harrington et al. 2013

Epipedobates tricolor

de Sá and Hill 1998

Harrington et al. 2013

Fejervarya cancrivora

Harrington et al. 2013

Hamptophryne boliviana

Harrington et al. 2013

Hyla versicolor

Harrington et al. 2013

Hylorina sylvatica

Harrington et al. 2013

Hymenochirus boettgeri

de Sá and Swart 1999

Hypsiboas lanciformis

de Sá 1988

Kassina senegalensis

Harrington et al. 2013

Leptodactylus chaquensis

Harrington et al. 2013

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Taxa

Standardization method (data type used)

Ontogenetic stages

Snout-vent length

(mm)

Ossification sequence

position

Osteopilus septentrionalis

Trueb 1966

Palaeobatrachus sp.

Harrington et al. 2013

Pelobates cultripes

Harrington et al. 2013

Philautus silus

Harrington et al. 2013

Phyllomedusa vaillanti

Harrington et al. 2013

Pipa myersi

Yeh 2002

Pipa pipa

Trueb et al. 2000

Harrington et al. 2013

Pseudacris regilla

Harrington et al. 2013

Pseudacris triseriata

Harrington et al. 2013

Pseudis platensis

Harrington et al. 2013

Pseudophryne bibronii

Harrington et al. 2013

Pyxicephalus adspersus

Harrington et al. 2013

Rana (Amerana) aurora

Harrington et al. 2013

Rana (Amerana) cascadae

Harrington et al. 2013

Rana (Amerana) pretiosa

Harrington et al. 2013

Rana (Rana) temporaria

Harrington et al. 2013

Rana (Pantherana) pipiens

Kemp and Hoyt 1969

Rhinophrynus dorsalis

Harrington et al. 2013

Shomronella jordanica

Harrington et al. 2013

Smilisca baudini

Harrington et al. 2013

Spea bombifrons

Wiens 1989

Spea multiplicata

Harrington et al. 2013

Triprion petasatus

Harrington et al. 2013

Uperoleia laevigata

Harrington et al. 2013

Xenopus laevis

Harrington et al. 2013

Mammalia

Bradypus variegatus

Hautier et al. 2011

Cavia porcellus

Hautier et al. 2013

Choloepus didactylus

Hautier et al. 2011

Cryptotis parva

Koyabu et al. 2011

Cyclopes didactylus

Hautier et al. 2011

Dasypus novemcinctus

Hautier et al. 2011

Dasyurus viverrinus

Hautier et al. 2013

Didelphis albiventris

de Oliveira et al. 1998

Echinops telfairi

Werneburg et al. 2013

Elephantulus rozeti

Hautier et al. 2013

Eremitalpa granti

Hautier et al. 2013

Erinaceus amurensis

Koyabu et al. 2011

Felis silvestris

Sánchez-Villagra et al. 2008

Homo sapiens

Hautier et al. 2013

Heterohyrax brucei

Hautier et al. 2013

Loxodonta africana

Hautier et al. 2012

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Taxa

Standardization method (data type used)

Ontogenetic stages

Snout-vent length

(mm)

Ossification sequence

position

Macropus eugenii

Hautier et al. 2013

Macroscelides proboscideus

Hautier et al. 2013

Manis javanica

Hautier et al. 2013

Meriones unguiculatus

Yukawa et al. 1999

Mesocricetus auratus

Hautier et al. 2013

Mogera wogura

Koyabu et al. 2011

Monodelphis domestica

Hautier et al. 2013

Mus musculus

Hautier et al. 2013

Ornithorhynchus anatinus

Weisbecker 2011

Orycteropus afer

Hautier et al. 2013

Perameles nasuta

Hautier et al. 2013

Peromyscus melanophrys

Hautier et al. 2013

Procavia capensis

Hautier et al. 2013

Rattus norvegicus

Hautier et al. 2013

Rhabdomys pumilio

Hautier et al. 2013

Rousettus amplexicaudatus

Hautier et al. 2013

Sus scrofa

Hautier et al. 2013

Tachyglossus aculeatus

Weisbecker 2011

Talpa spp.

Sánchez-Villagra et al. 2008

Tenrec ecaudatus

Werneburg et al. 2013

Tamandua tetradactyla

Hautier et al. 2011

Tarsius spectrum

Hautier et al. 2013

Trichosurus vulpecula

Weisbecker et al. 2008

Hautier et al. 2013

Tupaia javanica

Hautier et al. 2013

Squamata

Lacerta vivipara

Hautier et al. 2013

Lerista bougainvillii

Hugi et al. 2012

Liopholis whitii

Hugi et al. 2012

Hemiergis peronii

Hugi et al. 2012

Saiphos equalis

Hugi et al. 2012

Crocodylia

Alligator mississipiensis

Rieppel 1993a

Aves

Anas platyrhynchos

Maxwell et al. 2010

Cairina moschata

Maxwell et al. 2010

Coturnix coturnix

Maxwell et al. 2010

Coturnix coturnix (N&T)

Maxwell et al. 2010

Dromaius novaehollandiae

Maxwell et al. 2010

Dromaius novaehollandiae (YPM)

Maxwell et al. 2010

Gallus gallus

Maxwell et al. 2010

(which was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.

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Taxa

Standardization method (data type used)

Ontogenetic stages

Snout-vent length

(mm)

Ossification sequence

position

Gallus gallus (S&W)

Maxwell et al. 2010

Larus argentatus

Maxwell et al. 2010

Larus canus

Maxwell et al. 2010

Larus ridibundus

Maxwell et al. 2010

Meleagris gallopavo

Maxwell et al. 2010

Phalacrocorax auritus

Maxwell et al. 2010

Somateria mollissima

Maxwell et al. 2010

Stercorarius skua

Maxwell et al. 2010

Sterna hirundo

Maxwell et al. 2010

Struthio camelus

Maxwell et al. 2010

Testudines

Apalone spinifera

Sánchez-Villagra et al. 2008

Chelydra serpentina

Rieppel 1993b

Rieppel 1990, 1993b

Rieppel 1993b

Macrochelys temminckii

Sánchez-Villagra et al. 2008

Pelodiscus sinensis

Sánchez-Villagra et al. 2008

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