15.15: Evolution of Amniotes - Biology

15.15: Evolution of Amniotes - Biology

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Learning Outcomes

  • Discuss the evolution of amniotes

The first amniotes evolved from amphibian ancestors approximately 340 million years ago during the Carboniferous period. Synapsids include all mammals, including extinct mammalian species. Sauropsids include reptiles and birds, and can be further divided into anapsids and diapsids. The key differences between the synapsids, anapsids, and diapsids are the structures of the skull and the number of temporal fenestrae behind each eye (Figure 1).

Temporal fenestrae are post-orbital openings in the skull that allow muscles to expand and lengthen. Anapsids have no temporal fenestrae, synapsids have one, and diapsids have two. Anapsids include extinct organisms and may, based on anatomy, include turtles. However, this is still controversial, and turtles are sometimes classified as diapsids based on molecular evidence. The diapsids include birds and all other living and extinct reptiles.

The diapsids diverged into two groups, the Archosauromorpha (“ancient lizard form”) and the Lepidosauromorpha (“scaly lizard form”) during the Mesozoic period (Figure 2). The lepidosaurs include modern lizards, snakes, and tuataras. The archosaurs include modern crocodiles and alligators, and the extinct pterosaurs (“winged lizard”) and dinosaurs (“terrible lizard”). Clade Dinosauria includes birds, which evolved from a branch of dinosaurs.

Practice Question

Members of the order Testudines have an anapsid-like skull with one opening. However, molecular studies indicate that turtles descended from a diapsid ancestor. Why might this be the case?

[practice-area rows=”2″][/practice-area]
[reveal-answer q=”540837″]Show Answer[/reveal-answer]
[hidden-answer a=”540837″]The ancestor of modern Testudines may at one time have had a second opening in the skull, but over time this might have been lost.[/hidden-answer]

Evolution of large males is associated with female-skewed adult sex ratios in amniotes

Body size often differs between the sexes (leading to sexual size dimorphism, SSD), as a consequence of differential responses by males and females to selection pressures. Adult sex ratio (ASR, the proportion of males in the adult population) should influence SSD because ASR relates to both the number of competitors and available mates, which shape the intensity of mating competition and thereby promotes SSD evolution. However, whether ASR correlates with SSD variation among species has not been yet tested across a broad range of taxa. Using phylogenetic comparative analyses of 462 amniotes (i.e., reptiles, birds, and mammals), we fill this knowledge gap by showing that male bias in SSD increases with increasingly female-skewed ASRs in both mammals and birds. This relationship is not explained by the higher mortality of the larger sex because SSD is not associated with sex differences in either juvenile or adult mortality. Phylogenetic path analysis indicates that higher mortality in one sex leads to skewed ASR, which in turn may generate selection for SSD biased toward the rare sex. Taken together, our findings provide evidence that skewed ASRs in amniote populations can result in the rarer sex evolving large size to capitalize on enhanced mating opportunities.

Table S1.Relationship between SSD, ASR, and sex-biased mortalities in reptiles, using estimated body mass data for SSD calculation.

Table S2.Relationship between SSD, ASR, and sex-biased mortalities in reptiles, using body length data for SSD calculation.

Table S3.Relationship between SSD, ASR, and sex-biased mortalities in birds.

Table S4.Relationship between SSD, ASR, and sex-biased mortalities in mammals.

Table S5.Sensitivity analyses of the relationship between sexual size dimorphism and adult sex ratio.

Table S6.Analyses of the relationship between sexual size dimorphism and adult sex ratio with branch lengths calculated by different methods for the phylogeny used in the PGLS models.

Table S7.Analyses of the relationship between sexual size dimorphism and adult sex ratio in socially monogamous and socially polygamous species.

Table S8.Results of the phylogenetic path analyses using the R package “phylopath.”

Table S9.Phylogenetic path models using data of birds and mammals (i.e. excluding reptiles).

Figure S1.Sexual size dimorphism in relation to adult sex ratio in reptiles, birds and mammals.

Appendix 2. Parameters of the allometric equations for calculating body mass in reptiles.

Appendix 3. Methodological notes on path analyses applied to comparative data, and additional path analyses.

Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.

Evolution of Cortical Neurogenesis in Amniotes Controlled by Robo Signaling Levels

Cerebral cortex size differs dramatically between reptiles, birds, and mammals, owing to developmental differences in neuron production. In mammals, signaling pathways regulating neurogenesis have been identified, but genetic differences behind their evolution across amniotes remain unknown. We show that direct neurogenesis from radial glia cells, with limited neuron production, dominates the avian, reptilian, and mammalian paleocortex, whereas in the evolutionarily recent mammalian neocortex, most neurogenesis is indirect via basal progenitors. Gain- and loss-of-function experiments in mouse, chick, and snake embryos and in human cerebral organoids demonstrate that high Slit/Robo and low Dll1 signaling, via Jag1 and Jag2, are necessary and sufficient to drive direct neurogenesis. Attenuating Robo signaling and enhancing Dll1 in snakes and birds recapitulates the formation of basal progenitors and promotes indirect neurogenesis. Our study identifies modulation in activity levels of conserved signaling pathways as a primary mechanism driving the expansion and increased complexity of the mammalian neocortex during amniote evolution.

Keywords: Notch Pax6 Tbr2 electroporation evolution intermediate progenitor microcephaly radial glia.

Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.


Early Neurogenesis Is More Abundant…

Early Neurogenesis Is More Abundant in OB than Adjacent NCx in Mouse Embryo…

Differences between Early Growth of…

Differences between Early Growth of OB and NCx Correlate with Mitral Cell Generation,…

Higher Frequency of Direct Neurogenesis…

Higher Frequency of Direct Neurogenesis in OB than NCx (A–D) Distribution of apical…

Evidence Supporting Abundant Direct Neurogenesis…

Evidence Supporting Abundant Direct Neurogenesis in OB, but Not NCx, at E12.5, Related…

Videomicroscopy Analyses Demonstrating Abundant Direct…

Videomicroscopy Analyses Demonstrating Abundant Direct Neurogenesis in the OB, but Not NCx, at…

Robo1 and Robo2 Promote Direct…

Robo1 and Robo2 Promote Direct Neurogenesis in OB (A) ISH and qRT-PCR for…

Robo Receptors and Slit Ligands…

Robo Receptors and Slit Ligands Are Expressed in the Mouse Telencephalon during Embryonic…

Regulation of OB Neurogenesis and…

Regulation of OB Neurogenesis and Growth by Robo Receptors and Slit Ligands, Related…

Robo Receptors Cooperate with Dll1…

Robo Receptors Cooperate with Dll1 to Regulate Direct Neurogenesis in OB and NCx…

Functional Validation of Genetic Reagents…

Functional Validation of Genetic Reagents and Test of Interaction between Robo and Notch…

Robo/Dll1/Jag Signaling Drives Direct Genesis…

Robo/Dll1/Jag Signaling Drives Direct Genesis of Deep-Layer Corticofugal Neurons (A–G) ISH stains for…

Robo/Dll1 Signaling Drives Direct Genesis…

Robo/Dll1 Signaling Drives Direct Genesis of Deep-Layer Corticofugal Neurons and Regulates Direct Neurogenesis…

Robo/Dll1 Signaling Regulates the Balance…

Robo/Dll1 Signaling Regulates the Balance between Direct Neurogenesis and IPC Abundance in Chick…

Conserved Function of Robo/Dll1 in…

Conserved Function of Robo/Dll1 in Snake and Human Cortex to Regulate the Balance…

Evolution of Reptiles

Reptiles originated approximately 300 million years ago during the Carboniferous period. One of the oldest known amniotes is Casineria , which had both amphibian and reptilian characteristics. One of the earliest undisputed reptiles was Hylonomus . Soon after the first amniotes appeared, they diverged into three groups—synapsids, anapsids, and diapsids—during the Permian period. The Permian period also saw a second major divergence of diapsid reptiles into archosaurs (predecessors of crocodilians and dinosaurs) and lepidosaurs (predecessors of snakes and lizards). These groups remained inconspicuous until the Triassic period, when the archosaurs became the dominant terrestrial group due to the extinction of large-bodied anapsids and synapsids during the Permian-Triassic extinction. About 250 million years ago, archosaurs radiated into the dinosaurs and the pterosaurs.

Although they are sometimes mistakenly called dinosaurs, the pterosaurs were distinct from true dinosaurs ([Figure 4]). Pterosaurs had a number of adaptations that allowed for flight, including hollow bones (birds also exhibit hollow bones, a case of convergent evolution). Their wings were formed by membranes of skin that attached to the long, fourth finger of each arm and extended along the body to the legs.

Figure 4: Pterosaurs, which existed from the late Triassic to the Cretaceous period (210 to 65.5 million years ago), possessed wings but are not believed to have been capable of powered flight. Instead, they may have been able to soar after launching from cliffs. (credit: Mark Witton, Darren Naish)

The dinosaurs were a diverse group of terrestrial reptiles with more than 1,000 species identified to date. Paleontologists continue to discover new species of dinosaurs. Some dinosaurs were quadrupeds ([Figure 5]) others were bipeds. Some were carnivorous, whereas others were herbivorous. Dinosaurs laid eggs, and a number of nests containing fossilized eggs have been found. It is not known whether dinosaurs were endotherms or ectotherms. However, given that modern birds are endothermic, the dinosaurs that served as ancestors to birds likely were endothermic as well. Some fossil evidence exists for dinosaurian parental care, and comparative biology supports this hypothesis since the archosaur birds and crocodilians display parental care.

Figure 5: Edmontonia was an armored dinosaur that lived in the late Cretaceous period, 145.5 to 65.6 million years ago. (credit: Mariana Ruiz Villareal)

Dinosaurs dominated the Mesozoic Era, which was known as the “age of reptiles.” The dominance of dinosaurs lasted until the end of the Cretaceous, the last period of the Mesozoic Era. The Cretaceous-Tertiary extinction resulted in the loss of most of the large-bodied animals of the Mesozoic Era. Birds are the only living descendants of one of the major clades of dinosaurs.

Genomic innovations

As befits the taxonomic distinctiveness of the tuatara, we find that its genome displays multiple innovations in genes that are associated with immunity, odour reception, thermal regulation and selenium metabolism.

Genes of the major histocompatibility complex (MHC) have an important role in disease resistance, mate choice and kin recognition, and are among the most polymorphic genes in the vertebrate genome. Our annotation of MHC regions in the tuatara, and comparisons of the gene organization with that of six other species, identified 56 MHC genes (Extended Data Fig. 5, Supplementary Information 9).

Of the six comparison species, the genomic organization of tuatara MHC genes is most similar to that of the green anole, which we interpret as typical for Lepidosauria. Tuatara and other reptiles show a gene content and complexity more similar to the MHC regions of amphibians and mammals than to the highly reduced MHC of birds. Although the majority of genes annotated in the tuatara MHC are well-conserved as one-to-one orthologues, we observed extensive genomic rearrangements among these distant lineages.

The tuatara is a highly visual predator that is able to capture prey under conditions of extremely low light 2 . Despite the nocturnal visual adaptation of the tuatara, it shows strong morphological evidence of an ancestrally diurnal visual system 19 . We identified all five of the vertebrate visual opsin genes in the tuatara genome (Supplementary Information 10).

Our comparative analysis revealed one of the lowest rates of visual-gene loss known for any amniote, which contrasts sharply with the high rates of gene loss observed in ancestrally nocturnal lineages (Extended Data Fig. 6). Visual genes involved in phototransduction showed strong negative selection and no evidence for the long-term shifts in selective pressures that have been observed in other groups with evolutionarily modified photoreceptors 20 . The retention of five visual opsins and the conserved nature of the visual system also suggests tuatara possess robust colour vision, potentially at low light levels. This broad visual repertoire may be explained by the dichotomy in tuatara life history: juvenile tuatara often take up a diurnal and arboreal lifestyle to avoid the terrestrial, nocturnal adults that may predate them 2 . Collectively, these results suggest a unique path to nocturnal adaptation in tuatara from a diurnal ancestor.

Odorant receptors are expressed in the dendritic membranes of olfactory receptor neurons and enable the detection of odours. Species that depend strongly on their sense of smell to interact with their environment, find prey, identify kin and avoid predators may be expected to have a large number of odorant receptors. The tuatara genome contains 472 predicted odorant receptors, of which 341 sequences appear intact (Supplementary Information 11). The remainder lack the initial start codon, have frameshifts or are presumed to be pseudogenes. Many odorant receptors were found as tandem arrays, with up to 26 genes found on a single scaffold.

The number and diversity of odorant receptor genes varies greatly in Sauropsida: birds have 182–688 such genes, the green anole lizard has 156 genes, and crocodilians and testudines have 1,000–2,000 genes 21 . The tuatara has a number of odorant receptors similar to that of birds, but contains a high percentage of intact odorant receptor genes (85%) relative to published odorant receptor sets from the genomes of other sauropsids. This may reflect a strong reliance on olfaction by tuatara, and therefore pressure to maintain a substantial repertoire of odorant receptors (Extended Data Fig. 7). There is some evidence that olfaction has a role in identifying prey 2 , as well as suggestions that cloacal secretions may act as chemical signals.

The tuatara is a behavioural thermoregulator, and is notable for having the lowest optimal body temperature of any reptile (16–21 °C). Genes that encode transient receptor potential ion channels (TRP genes) have an important role in thermoregulation, as these channels participate in thermosensation and cardiovascular physiology 22 this led us to hypothesize that TRP genes may be linked to the thermal tolerance of the tuatara. Our comparative genomic analysis of TRP genes in the tuatara genome identified 37 TRP-like sequences, spanning all 7 known subfamilies of TRP genes (Extended Data Fig. 8, Supplementary Information 12)— an unusually large repertoire of TRP genes.

Among this suite of genes, we identified thermosensitive and non-thermosensitive TRP genes that appear to result from gene duplication, and have been differentially retained in the tuatara. For example, the tuatara is unusual in possessing an additional copy of a thermosensitive TRPV-like gene (TRPV1/2/4, sister to the genes TRPV1, TRPV2 and TRPV4) that has classically been linked to the detection of moderate-to-extreme heat 22 —a feature it shares with turtles. A strong signature of positive selection among heat-sensitive TRP genes (TRPA1, TRPM and TRPV) was also observed.

In general, these results show a high rate of differential retention and positive selection in genes for which a function in heat sensation is well-established 22 . It therefore seems probable that the genomic changes in TRP genes are associated with the evolution of thermoregulation in tuatara.

Barring tortoises, tuatara are the longest lived of the reptiles—probably exceeding 100 years of age 2 . This enhanced lifespan may be linked to genes that afford protection against reactive oxygen species. One class of gene products that affords such protection is the selenoproteins. The human genome encodes 25 selenoproteins, the roles of which include antioxidation, redox regulation, thyroid hormone synthesis and calcium signal transduction, among others 23 .

We identified 26 genes that encode selenoproteins in the tuatara genome, as well as 4 selenocysteine-specific tRNA genes all of these appear to be functional (Supplementary Information 13). Although further work is needed, the additional selenoprotein gene (relative to the human genome) and the selenocysteine-specific tRNA genes may be linked to the longevity of tuatara or might have arisen as a response to the low levels of selenium and other trace elements in the terrestrial systems of New Zealand.

Tuatara has a unique mode of temperature-dependent sex determination, in which higher temperatures during egg incubation result in males 2 . We found orthologues for many genes that are known to act antagonistically in masculinizing (for example, SF1 and SOX9) and feminizing (for example, RSPO1 and WNT4) gene networks to promote testicular or ovarian development, respectively 24 . We also found orthologues of several genes that have recently been implicated in temperature-dependent sex determination, including CIRBP 24 (Supplementary Information 17, Supplementary Table 17.2). Tuatara possess no obviously differentiable sex chromosomes 5 , and we found no significant sex-specific differences in global CG methylation (Fig. 3a) and no sex-specific single-nucleotide variants between male and female tuatara (Fig. 3b). On a gene-by-gene basis, sex-specific differences in methylation and gene expression patterns probably exist, but this remains to be investigated.

Accelerated evolution of an Lhx2 enhancer shapes mammalian social hierarchies

Social hierarchies emerged during evolution, and social rank influences behavior and health of individuals. However, the evolutionary mechanisms of social hierarchy are still unknown in amniotes. Here we developed a new method and performed a genome-wide screening for identifying regions with accelerated evolution in the ancestral lineage of placental mammals, where mammalian social hierarchies might have initially evolved. Then functional analyses were conducted for the most accelerated region designated as placental-accelerated sequence 1 (PAS1, P = 3.15 × 10 -18 ). Multiple pieces of evidence show that PAS1 is an enhancer of the transcription factor gene Lhx2 involved in brain development. PAS1s isolated from various amniotes showed different cis-regulatory activity in vitro, and affected the expression of Lhx2 differently in the nervous system of mouse embryos. PAS1 knock-out mice lack social stratification. PAS1 knock-in mouse models demonstrate that PAS1s determine the social dominance and subordinate of adult mice, and that social ranks could even be turned over by mutated PAS1. All homozygous mutant mice had normal huddled sleeping behavior, motor coordination and strength. Therefore, PAS1-Lhx2 modulates social hierarchies and is essential for establishing social stratification in amniotes, and positive Darwinian selection on PAS1 plays pivotal roles in the occurrence of mammalian social hierarchies.

Conflict of interest statement

The authors declare no competing interests.


Fig. 1. Genome-wide screening for PASs.

Fig. 1. Genome-wide screening for PASs.

Fig. 2. Effects of PAS1s from various…

Fig. 2. Effects of PAS1s from various amniotes on reporter gene expression in mouse neuroblastoma…

Fig. 3. Effect of PAS1s from various…

Fig. 3. Effect of PAS1s from various amniotes on the expression of Lhx2 in embryonic…

Fig. 4. Modulation of social hierarchy by…

Fig. 4. Modulation of social hierarchy by PAS1s from various amniotes in caged adult male…

Lungs of the first amniotes: why simple if they can be complex?

We show—in contrast to the traditional textbook contention—that the first amniote lungs were complex, multichambered organs and that the single-chambered lungs of lizards and snakes represent a secondarily simplified rather than the plesiomorphic condition. We combine comparative anatomical and embryological data and show that shared structural principles of multichamberedness are recognizable in amniotes including all lepidosaurian taxa. Sequential intrapulmonary branching observed during early organogenesis becomes obscured during subsequent growth, resulting in a secondarily simplified, functionally single-chambered lung in lepidosaurian adults. Simplification of pulmonary structure maximized the size of the smallest air spaces and eliminated biophysically compelling surface tension problems that were associated with miniaturization evident among stem lepidosaurmorphs. The remaining amniotes, however, retained the multichambered lungs, which allowed both large surface area and high pulmonary compliance, thus initially providing a strong selective advantage for efficient respiration in terrestrial environments. Branched, multichambered lungs instead of simple, sac-like organs were part and parcel of the respiratory apparatus of the first amniotes and pivotal for their success on dry land, with the sky literally as the limit.

1. Introduction

Adaptations in the respiratory system were required to ensure success in colonizing dry land and in the evolution of high aerobic performance and active flight [1]. While particularly in tetrapods the main organs for aerobic gas exchange are lungs, their evolutionary origin can be traced back to fish-like ancestors [1]. In amniotes, the primarily fully terrestrial vertebrates, lungs are the principle sites for air breathing and their anatomy exhibits tremendous structural diversity. It ranges from simple, single-chambered organs to highly complex, multichambered and branched lungs, as well as transitional types covering almost everything in between [2,3]. Although aspiration breathing is an essential character of the amniote condition [2], it is commonly assumed, based on the presence of single-chambered lungs in the majority of lepidosaurs, that the rather simple organs observed in extant amphibians also represent the ancestral amniote gas exchanger [4–7]. This combination of an effective breathing mechanism with simple lungs represents a paradox for basal amniotes. It further suggests that the complex, multichambered lungs of mammals, crocodiles/birds, turtles and monitor lizards each evolved independently as an exit from this paradox [1,2]. The present study addresses the question whether the latter scenario can be supported or if a common Bauplan of amniote lungs other than that of a simple ‘sac’ exists.

2. Material and methods

We studied the lungs of 187 specimens, representing 73 species from 42 different ‘families’, covering all major tetrapod lineages. Lungs were excised, dehydrated and dried or injected with silicone elastomer for macroscopic examination. A tuatara lung was scanned with a v|tome|x s µCT device (phoenix|X-ray) at the Steinmann-Institut, Universität Bonn (Germany) with a resolution of 69.57 µm per voxel and visualized using VGS tudio MAX v. 2.0 (Volume Graphics). Embryos of the Madagascar ground gecko, Paroedura picta, at different developmental stages, fixed in 4% paraformaldehyde/phosphate-buffered saline (PBS), were dissected under a stereoscopic microscope and their lungs were removed. Lungs were analysed in PBS and imaged using an EOS 5D Mark II mounted on a Zeiss TESSOVAR with a Schott KL 1500 illuminant adjusted to a spectral regime of 540 nm. Ancestral state reconstructions were performed using parsimony and unordered character states in M esquite v. 2.74 [8]. See the electronic supplementary material for full methods.

3. Results and discussion

The lungs of lepidosaurs have in common with the more complex amniote lungs a subapical bronchial entrance, near the point where the pulmonary artery enters the lung [9]. In amphibians, the entrance to the lung is apical (figure 1a), as is also the case for the pulmonary artery (electronic supplementary material, figure S1a). In multichambered lungs, the pulmonary artery follows the major branches of airways within the lung [10,11]. In single-chambered and transitional type lepidosaurian lungs, the arterial branching pattern is similar, in that arterial branches hierarchically supply large internal partitions that frequently extend into the lung lumen as small septa (figure 1b). Even in the highly derived lungs of cobras (figure 1d), this strictly hierarchical branching pattern of the blood vessels is evident (figure 1e). The lungs of the only extant non-squamate lepidosaur, the tuatara (Sphenodon punctatus), which previously have been regarded as more amphibian-like than lizard-like [12], are here of critical importance. Externally, they closely resemble typical single-chambered lizard lungs (figure 1f). But also internally we have identified a row of septa, particularly in the ventral region (figure 1g,h), that reflects the course of the major branches of the pulmonary artery (figure 1f). This sequential structural pattern completely agrees with that in other lepidosaurs (electronic supplementary material, figure S1b). Thus, there are pulmonary and vascular indicators for a hierarchical subdivision in the single-chambered lungs of all lepidosaurs.

Figure 1. Pulmonary diversity in tetrapods. (a) Even the largest amphibians possess single-chambered lungs with an apical entrance. (b) Amniotes exhibit a subapical bronchial entrance and a variety of internal structuralization. Large septa can separate chambers, which, in turn, are partially subdivided by smaller septa (Se), exactly followed by branches of the pulmonary artery (Pa). (c) The subapical bronchial entrance (*) is seen even in the vestigial left lung of cobras. (d,e) There, only the right lung (Rlu) develops to a functional organ. These lungs are remarkably tube-like and exhibit tissue for gas exchange (Get) only immediately posterior to the bronchial entrance (Be), which more or less continuously follows the trachea (Tr), while the more distal portion is characterized as a sac-like region (sac, only partially shown). Despite this fundamentally single-chambered appearance, not only is the bronchial entrance subapical, but also the pulmonary artery (Pa) exhibits the strict hierarchical branching pattern typical of amniote lungs. (fh) The tuatara exhibits all these traits. (i) Embryonic lungs of Madagascar ground geckos show a dorsal bud that makes the bronchial entrance subapical. (j) Later in ontogeny, additional buds form an almost multichambered lung. (km) In the expansion phase, the lung dilates, obscuring the initial multichamberedness.

Geckos represent a basal radiation of lizards with single-chambered lungs [3,13] and thus constitute an ideal model for developmental studies on these structural vestiges of putative ‘multichamberedness’. In P. picta, the lung Anlagen show a clear sequential budding of dorsal and ventral chamber-like elements during early developmental stages (figure 1i,j). In this ‘branching phase’, the most anterior element becomes enlarged and extends craniad, causing the bronchial entrance to become subapical (figure 1j). During the ensuing ‘expansion phase’, the central pulmonary lumen becomes dilated, obscuring the sequential structure, which can be identified in the adult only by small septa, each with individual vascular supply (figure 1km electronic supplementary material, figure S2). The early stages are strikingly similar to the sequentially branched structure of developing mammalian [14], chelonian [15], crocodylian [16] and avian lungs [17] (electronic supplementary material, figure S1c) and are also virtually identical with the Bauplan of monitor lungs (electronic supplementary material, figure S3). Interestingly, the vestigial left lung of cobras exhibits a remarkable similarity to the gecko's lungs during early ontogeny, including the subapical bronchial entrance (figure 1c). Such a subapical bronchial entrance, as well as sequential internal subdivision during early organogenesis, appears not yet to have been observed in amphibians. Furthermore, no blood vessel pattern that betrays a transient subapical bronchial entrance exists in either embryonic or adult lungs of the species studied (electronic supplementary material, figure S1a). Such a rather simple internal Bauplan even applies to the largest extant amphibians: e.g. the Japanese giant salamander (Andrias japonicus) (figure 1a).

We suggest that a common genetic programme for intrapulmonary branching, acting during early ontogenetic stages, is shared at least by all amniotes and represents a fertile ground for further research. Ancestral state reconstructions support the plesiomorphy of the amniote pattern described here (see electronic supplementary material, figures S4 and S5). Thus, the lepidosaurs must have undergone a secondary simplification of their pulmonary Bauplan rather than inheriting an undivided and unbranched lung structure from their ancestors.

As multichambered lungs can provide greater surface areas and higher pulmonary compliances than single-chambered lungs of equivalent volume, they are more cost-effective to ventilate [18,19]. Thus, an evolutionary scenario whereby single-chambered lungs represent a more highly derived condition raises the question whether this simplification presented some selective advantage, or multichambered lungs presented a disadvantage. A strong disadvantage would have been present if lepidosaurs evolved from miniscule ancestors. In fact, the Mesozoic fossil record of stem lepidosauromorphs shows that species with a body cavity length of less than 3 cm, comparable with the smallest extant lizards, actually existed [20,21]. A respective miniaturization of a multichambered lung would result in extremely tiny air spaces. As surface tension increases dramatically with a decrease in diameter, the result would be low-compliance lungs that are extremely difficult to inflate without an auxiliary breathing structure [18,19]. Mammals and birds of such small size (alveolar diameter approx. 50 µm and air capillaries of 3 µm, respectively) compensate for these biophysical constraints through a variety of auxiliary structures, such as the diaphragm or the air sacs, respectively [1]. In newborn, extremely tiny marsupials, the lungs are very immature and require substantial postnatal development to become functional, while oxygen is supplied via cutaneous gas exchange [22]. The majority of lepidosaurs, however, evolved a functionally single-chambered lung, whereby the ancestral branched nature is evident only during early development, when the lungs are still fluid-filled and non-functional.

In this context, it is of particular interest that varanoid lizards are highly derived squamates [13] and that they exhibit very complex and multichambered lungs [3]. We hypothesize that basal representatives of the varanoid lineage exploited the ‘multichamberedness’ of lung Anlagen retained from the ancestral amniote condition to ‘reinvent’ a multichambered lung (see electronic supplementary material, figure S3).

In summary, single-chambered lungs of lizards and snakes reveal the same basic ontogenetic amniote pattern observed in more complex, multichambered ones. In spite of their extreme complexity, mammalian lungs display an underlying developmental sequence that can be explained by only three different types of branching [14]. Findings from turtles [23] and archosaurs [10,17], where the pulmonary Bauplan follows a similar strict hierarchical ontogenetic sequence, as well as the data for lepidosaurs presented here, indicate that deep homologous genetic processes are involved in these diverse lineages. Taken together, the evidence integrated here is consistent with a scenario for amniote lung evolution (figure 2) that starts with complexity as a key to terrestrialization.

Figure 2. Pulmonary evolution in amniotes. In amphibians, lungs are single-chambered and initially only complement gills and skin (a). In amniotes, the lungs are the principle site for gas exchange multichamberedness is shared by all amniotes and was a key for conquering dry land (b). In mammals, pronounced intrapulmonary branching led to the bronchioalveolar lung (c) turtle lungs approximate the plesiomorphic amniote condition (d). Archosaurs (crocodiles, birds) retained a multichambered lung, and birds evolved air sacs and the parabronchial lung (e). The ancestors of lepidosaurs underwent miniaturization, preventing them from retaining a multichambered lung. Multichamberedness is still ontogenetically visible, despite the adult single-chamberedness (f).

Morphological research on amniote eggs and embryos: An introduction and historical retrospective

Daniel G. Blackburn, Department of Biology, Trinity College, Hartford, CT 06106 USA.

Contribution: Conceptualization, Writing - original draft, Writing - review & editing

Department of Biological Sciences, East Tennessee State University, Johnson City, Tennessee, USA

Contribution: Conceptualization, Writing - review & editing

Department of Biology and Electron Microscopy Center, Trinity College, Hartford, Connecticut, USA

Daniel G. Blackburn, Department of Biology, Trinity College, Hartford, CT 06106 USA.

Contribution: Conceptualization, Writing - original draft, Writing - review & editing

Department of Biological Sciences, East Tennessee State University, Johnson City, Tennessee, USA

Contribution: Conceptualization, Writing - review & editing


Evolution of the terrestrial egg of amniotes (reptiles, birds, and mammals) is often considered to be one of the most significant events in vertebrate history. Presence of an eggshell, fetal membranes, and a sizeable yolk allowed this egg to develop on land and hatch out well-developed, terrestrial offspring. For centuries, morphologically-based studies have provided valuable information about the eggs of amniotes and the embryos that develop from them. This review explores the history of such investigations, as a contribution to this special issue of Journal of Morphology, titled Developmental Morphology and Evolution of Amniote Eggs and Embryos. Anatomically-based investigations are surveyed from the ancient Greeks through the Scientific Revolution, followed by the 19th and early 20th centuries, with a focus on major findings of historical figures who have contributed significantly to our knowledge. Recent research on various aspects of amniote eggs is summarized, including gastrulation, egg shape and eggshell morphology, eggs of Mesozoic dinosaurs, sauropsid yolk sacs, squamate placentation, embryogenesis, and the phylotypic phase of embryonic development. As documented in this review, studies on amniote eggs and embryos have relied heavily on morphological approaches in order to answer functional and evolutionary questions.


The tree of life is rapidly coming into focus as hundreds of molecular phylogenies are published each year. For the most part, trees from morphology and molecules have agreed, but there are some notable exceptions, with one being the position of turtles. Classically, the absence of temporal openings in the skull of turtles, the anapsid condition, has been used as evidence to place turtles at the bottom of the amniote tree, after the single-holed (synapsid) mammals split off but before the double-holed (diapsid) reptiles diversified [1] (Figure 1a). Those diapsids include the lepidosaurs (lizards, snakes, amphisbaenians, and tuataras), crocodilians, and birds. Some morphologists have agreed with the classical position of turtles [2] whereas others have interpreted data differently, finding that turtles group with lepidosaurs [3] (Figure 1b). In contrast, virtually all molecular studies, including a recent one in this journal, have found turtles to group with birds and crocodilians, the archosaurs [4–7] (Figure 1c). Although a few morphological characters support a turtle-archosaur group [8], morphologists in general have not embraced the molecular tree.

The three competing theories for the evolutionary position of turtles among amniote vertebrates. (a) Morphology-1 (turtles early): the classic morphological hypothesis, with turtles branching early. (b) Morphology-2 (turtles with lepidosaurs): another morphological hypothesis, which groups turtles with lizards and their relatives. (c) Molecules (turtles with archosaurs): the molecular hypothesis, which groups turtles with birds and crocodilians.

This dispute is similar to other major controversies in amniote evolution, such as the relationships of squamate reptiles (lizards, snakes, and amphisbaenians) [9] and African mammals (Afrotheria) [10]. However, resolution of those controversies has varied. For example, the molecular tree of squamates has some support from morphological characters, and is slowly gaining hold among evolutionary biologists, but not without resistance. Few morphological characters support Afrotheria, but the inferred biogeographic story, involving continental breakup in the Cretaceous, is so compelling that it quickly gained wide acceptance. This suggests that corroboration from independent evidence, such as biogeography, can go a long way toward resolving a conflict. However, no broad consensus has emerged in the case of turtles. Morphological data and molecular data remain at odds, and biogeographic support for the position of turtles is lacking, probably because turtles originated on the supercontinent Pangaea, before it broke apart. The position of turtles, therefore, represents a classic example of conflict between molecules and morphology.

The study of Chiari et al. [7] raises the molecular bar even higher. They analyzed DNA sequence data from 248 genes in 14 amniotes, subjecting those data to a battery of phylogenetic analyses designed to overcome potential biases. The result was, once again, significant support for a close relationship of turtles and archosaurs. A separate study published last year, involving thousands of genes from the transcriptome but fewer species, obtained the same result [6]. These two studies are the largest yet to address the higher-level relationships of amniotes and demonstrate that the molecular position of turtles (Figure 1c) is unwavering.

In systematics there is usually a trade-off between the number of characters (for example, nucleotide sites) and taxa (for example, species), and even these two large molecular studies could be viewed as having too few taxa. The authors of both studies encountered some systematic biases during their analyses, likely attributable to limited taxonomic sampling. This means that there is room for improvement in the future. For example, the earliest-branching lepidosaurs (tuataras) and birds (paleognaths) would be important additions to large molecular data sets, to help stabilize the phylogenies.

One molecular study using micro-RNAs, published recently, stands out among all others in concluding that turtles are most closely related to lepidosaurs, thus supporting morphology [11]. At face value, this could be viewed as reconciling the lengthy dispute in favor of morphology, or at least muddying the waters. But on closer inspection, only a single character supports two key nodes in that tree (reptiles and archosaurs), making the result non-significant and thus not a challenge to the molecular position of turtles. The usefulness of micro-RNAs for tree-building has yet to be established.

On the morphological side, there is mostly agreement that turtles are diapsids [8], albeit closer to lepidosaurs (Figure 1b) than to archosaurs, which is in better agreement with molecules than the classical position. Also, the classical position (Figure 1a) requires a lengthy gap in the fossil record leading to turtles, which is hard to explain for a vertebrate group that is known to fossilize well. In either case, the morphological evidence cannot be dismissed because it is critical for understanding relationships of the major groups of amniotes, such as pareiasaurs, aetosaurs, and ichthyosaurs, among others, that are long extinct. Moving turtles to another location in the tree, with archosaurs, would cause reinterpretations of character evolution, probably impacting the evolutionary position of extinct groups.

Fortunately, major conflicts between molecules and morphology, like this one involving turtles, are not common in evolutionary biology. They are usually resolved relatively quickly as new data are collected and analyzed, or independent evidence such as biogeography is brought to bear on the issue. In this case, determining which result is correct will require additional evidence, such as greater taxon-sampling in molecular data sets and greater scrutiny of the morphological and fossil evidence. In the end, having a stable amniote tree that includes turtles and fossil groups will be of tremendous value in understanding the ecological, physiological, and biogeographic history of amniotes and their adaptive radiation on land and in the seas.

15.15: Evolution of Amniotes - Biology

The first amniotes evolved from amphibian ancestors approximately 340 million years ago during the Carboniferous period. The early amniotes diverged into two main lines soon after the first amniotes arose. The initial split was into synapsids and sauropsids. Synapsids include all mammals, including extinct mammalian species. Synapsids also include therapsids, which were mammal-like reptiles from which mammals evolved. Sauropsids include reptiles and birds, and can be further divided into anapsids and diapsids. The key differences between the synapsids, anapsids, and diapsids are the structures of the skull and the number of temporal fenestrae behind each eye (Figure 1).

Figure 1. Compare the skulls and temporal fenestrae of anapsids, synapsids, and diapsids. Anapsids have no openings, synapsids have one opening, and diapsids have two openings.

Temporal fenestrae are post-orbital openings in the skull that allow muscles to expand and lengthen. Anapsids have no temporal fenestrae, synapsids have one, and diapsids have two. Anapsids include extinct organisms and may, based on anatomy, include turtles. However, this is still controversial, and turtles are sometimes classified as diapsids based on molecular evidence. The diapsids include birds and all other living and extinct reptiles.

The diapsids diverged into two groups, the Archosauromorpha (“ancient lizard form”) and the Lepidosauromorpha (“scaly lizard form”) during the Mesozoic period (Figure 2). The lepidosaurs include modern lizards, snakes, and tuataras. The archosaurs include modern crocodiles and alligators, and the extinct pterosaurs (“winged lizard”) and dinosaurs (“terrible lizard”). Clade Dinosauria includes birds, which evolved from a branch of dinosaurs.

Figure 2. This chart shows the evolution of amniotes. The placement of Testudines (turtles) is currently still debated.

Practice Question

Members of the order Testudines have an anapsid-like skull with one opening. However, molecular studies indicate that turtles descended from a diapsid ancestor. Why might this be the case?

Watch the video: Evolution of Early Reptiles (August 2022).