Cysteine Enrichment Mediates Co-Option of Uricase in Reptilian Skin and Transition to Uricotelism.

Uric acid is the main means of nitrogen excretion in uricotelic vertebrates (birds and reptiles) and the end product of purine catabolism in humans and a few other mammals. While uricase is inactivated in mammals unable to degrade urate, the presence of orthologous genes without inactivating mutations in avian and reptilian genomes is unexplained. Here we show that the Gallus gallus gene we name cysteine-rich urate oxidase (CRUOX) encodes a functional protein representing a unique case of cysteine enrichment in the evolution of vertebrate orthologous genes. CRUOX retains the ability to catalyze urate oxidation to hydrogen peroxide and 5-hydroxyisourate (HIU), albeit with a 100-fold reduced efficiency. However, differently from all uricases hitherto characterized, it can also facilitate urate regeneration from HIU, a catalytic property that we propose depends on its enrichment in cysteine residues. X-ray structural analysis highlights differences in the active site compared to known orthologs and suggests a mechanism for cysteine-mediated self-aggregation under H2O2-oxidative conditions. Cysteine enrichment was concurrent with the transition to uricotelism and a shift in gene expression from the liver to the skin where CRUOX is co-expressed with ß-keratins. Therefore, the loss of urate degradation in amniotes has followed opposite evolutionary trajectories: while uricase has been eliminated by pseudogenization in some mammals, it has been repurposed as a redox-sensitive enzyme in the reptilian skin.

Zona pellucida family genes in Chinese pond turtle: identification, expression profiles, and role in the spermatozoa acrosome reaction†.

The zona pellucida (ZP) is an extracellular matrix that surrounds all vertebrate eggs, and it is involved in fertilization and species-specific recognition. Numerous in-depth studies of the ZP proteins of mammals, birds, amphibians, and fishes have been conducted, but systematic investigation of the ZP family genes and their role during fertilization in reptiles has not been reported to date. In this study, we identified six turtle ZP (Tu-ZP) gene subfamilies (Tu-ZP1, Tu-ZP2, Tu-ZP3, Tu-ZP4, Tu-ZPD, and Tu-ZPAX) based on whole genome sequence data from Mauremys reevesii. We found that Tu-ZP4 had large segmental duplication and was distributed on three chromosomes, and we also detected gene duplication in the other Tu-ZP genes. To evaluate the role of Tu-ZP proteins in sperm-egg binding, we assessed the expression pattern of these Tu-ZP proteins and their ability to induce the spermatozoa acrosome reaction in M. reevesii. In conclusion, this is the first report of the existence of gene duplication of Tu-ZP genes and that Tu-ZP2, Tu-ZP3, and Tu-ZPD can induce acrosome exocytosis of spermatogenesis in the reptile.

Cloacal virome of an ancient host lineage - The tuatara (Sphenodon punctatus) - Reveals abundant and diverse diet-related viruses.

Tuatara (Sphenodon punctatus) are one of the most phylogenetically isolated vertebrate species and provide a unique host system to study virus evolution. While the tuatara genome, sequenced in 2020, revealed many endogenous viral elements, we know little of the exogenous viruses that infect tuatara. We performed a metatranscriptomics study of tuatara cloaca samples from a wild population on Takapourewa (Stephens Island), Aotearoa New Zealand. From these data we identified 49 potentially novel viral species that spanned 19 RNA viral families and/or orders, the vast majority (48) of which were likely dietary-related. Notably, using a protein structure homology search, we identified a highly divergent novel virus within the Picornaviridae which may directly infect tuatara. Additionally, two endogenous tuatara adintoviruses were characterised that exhibited long-term viral-host co-divergence. Overall, our results indicate that the tuatara cloacal virome is highly diverse, likely due to a large number of dietary-related viruses.

Keratinization and Cornification are not equivalent processes but keratinization in fish and amphibians evolved into cornification in terrestrial vertebrates.

The present account offers a generalized view of the evolution of process of terminal differentiation in keratinocytes of the epidermis in anamniotes, indicated as keratinization, into a further differentiating process of cornification in the skin and appendages of terrestrial vertebrates. Keratinization indicates the prevalent accumulation of intermediate filaments of keratins (IFKs) and is present in most fish and amphibian epidermis and inner epithelia of all vertebrates. During land adaptation, terrestrial vertebrates evolved a process of cornification and keratinocytes became dead corneocytes by the addition of numerous others proteins to the IFKs framework, represented by keratin-associated proteins (KAPs) and corneous proteins (CPs). Most of genes coding for these types of proteins are localized in chromosomal loci different and un-related from those of IFKs, and CPs originated from a gene cluster indicated as epidermal differentiation complex. During the evolution of reptiles and birds, the epidermis and corneous derivatives such as scales, claws, beaks and feathers mainly accumulate a type of CPs that overcome IFKs and containing a 34 amino acid beta-sheet core indicated as corneous beta proteins, formerly known as beta-keratins. Mammals did not evolve a beta-sheet core in their CPs and KAPs but instead produced numerous cysteine-rich IFKs in their epidermis and specialized KAPs in hairs, claws, nails, hooves and horns.

Keratin intermediate filament chains in tuatara (Sphenodon punctatus): A comparison of tuatara and human sequences.

Determination of the sequences of the keratin intermediate filament chains in tuatara has shown that these are closely akin to the α-keratins in human and other vertebrates, especially in the central, coiled-coil rod region. The domain lengths within the rod are preserved exactly, both Type I and Type II chains have been recognised, and sequence identity/homology exists between their respective chains. Nonetheless, there are characteristic differences in amino acid composition and sequence between their respective head (N-terminal) domains and their tail (C-terminal) domains, though some similarities are retained. Further, there is evidence of tandem repeats of a variety of lengths in the tuatara heads and tails indicative of sequence duplication events. These are not evident in human α-keratins and would therefore have implications for the physical attributes of the tissues in the two species. Multiple families of keratin-associated proteins that are ultra-high cysteine-rich or glycine + tyrosine-rich in human and other species do not have direct equivalents in the tuatara. Although high-sulphur proteins are present in tuatara the cysteine residue contents are significantly lower than in human. Further, no sequence homologies between the HS proteins in the two species have been found, thereby casting considerable doubt as to whether any matrix-forming high-sulphur proteins exist in tuatara. These observations may be correlated with the numerous cysteine-rich ß-keratins (corneous ß-proteins) that are present in tuatara, but which are conspicuously absent in mammals.

Development, structure, and protein composition of reptilian claws and hypotheses of their evolution.

Here, we review the development, morphology, genes, and proteins of claws in reptiles. Claws likely form owing to the inductive influence of phalangeal mesenchyme on the apical epidermis of developing digits, resulting in hyperproliferation and intense protein synthesis in the dorsal epidermis, which forms the unguis. The tip of claws results from prevalent cell proliferation and distal movement along most of the ungueal epidermis in comparison to the ventral surface forming the subunguis. Asymmetrical growth between the unguis and subunguis forces beta-cells from the unguis to rotate into the apical part of the subunguis, sharpening the claw tip. Further sharpening occurs by scratching and mechanical wearing. Ungueal keratinocytes elongate, form an intricate perimeter and cementing junctions, and remain united impeding desquamation. In contrast, thin keratinocytes in the subunguis form a smooth perimeter, accumulate less corneous beta proteins (CBPs) and cysteine-poor intermediate filament (IF)-keratins, and desquamate. In addition to prevalent glycine-cysteine-tyrosine rich CBPs, special cysteine-rich IF-keratins are also synthesized in the claw, generating numerous SS bonds that harden the thick and compact corneous material. Desquamation and mechanical wear at the tip ensure that the unguis curvature remains approximately stable over time. Reptilian claws are likely very ancient in evolution, although the unguis differentiated like the outer scale surface of scales, while the subunguis might have derived from the inner scale surface. The few hair-like IF-keratins synthesized in reptilian claws indicate that ancestors of sauropsids and mammals shared cysteine-rich IF-keratins. However, the number of these keratins remained low in reptiles, while new types of CBPs function to strengthen claws.

MeDAS: a Metazoan Developmental Alternative Splicing database.

Alternative splicing is widespread throughout eukaryotic genomes and greatly increases transcriptomic diversity. Many alternative isoforms have functional roles in developmental processes and are precisely temporally regulated. To facilitate the study of alternative splicing in a developmental context, we created MeDAS, a Metazoan Developmental Alternative Splicing database. MeDAS is an added-value resource that re-analyses publicly archived RNA-seq libraries to provide quantitative data on alternative splicing events as they vary across the time course of development. It has broad temporal and taxonomic scope and is intended to assist the user in identifying trends in alternative splicing throughout development. To create MeDAS, we re-analysed a curated set of 2232 Illumina polyA+ RNA-seq libraries that chart detailed time courses of embryonic and post-natal development across 18 species with a taxonomic range spanning the major metazoan lineages from Caenorhabditis elegans to human. MeDAS is freely available at https://das.chenlulab.com both as raw data tables and as an interactive browser allowing searches by species, tissue, or genomic feature (gene, transcript or exon ID and sequence). Results will provide details on alternative splicing events identified for the queried feature and can be visualised at the gene-, transcript- and exon-level as time courses of expression and inclusion levels, respectively.

Metabolic trait diversity shapes marine biogeography.

Climate and physiology shape biogeography, yet the range limits of species can rarely be ascribed to the quantitative traits of organisms1-3. Here we evaluate whether the geographical range boundaries of species coincide with ecophysiological limits to acquisition of aerobic energy4 for a global cross-section of the biodiversity of marine animals. We observe a tight correlation between the metabolic rate and the efficacy of oxygen supply, and between the temperature sensitivities of these traits, which suggests that marine animals are under strong selection for the tolerance of low O2 (hypoxia)5. The breadth of the resulting physiological tolerances of marine animals predicts a variety of geographical niches-from the tropics to high latitudes and from shallow to deep water-which better align with species distributions than do models based on either temperature or oxygen alone. For all studied species, thermal and hypoxic limits are substantially reduced by the energetic demands of ecological activity, a trait that varies similarly among marine and terrestrial taxa. Active temperature-dependent hypoxia thus links the biogeography of diverse marine species to fundamental energetic requirements that are shared across the animal kingdom.

Megaevolutionary dynamics and the timing of evolutionary innovation in reptiles.

The origin of phenotypic diversity among higher clades is one of the most fundamental topics in evolutionary biology. However, due to methodological challenges, few studies have assessed rates of evolution and phenotypic disparity across broad scales of time to understand the evolutionary dynamics behind the origin and early evolution of new clades. Here, we provide a total-evidence dating approach to this problem in diapsid reptiles. We find major chronological gaps between periods of high evolutionary rates (phenotypic and molecular) and expansion in phenotypic disparity in reptile evolution. Importantly, many instances of accelerated phenotypic evolution are detected at the origin of major clades and body plans, but not concurrent with previously proposed periods of adaptive radiation. Furthermore, strongly heterogenic rates of evolution mark the acquisition of similarly adapted functional types, and the origin of snakes is marked by the highest rates of phenotypic evolution in diapsid history.

Discovery of a New TLR Gene and Gene Expansion Event through Improved Desert Tortoise Genome Assembly with Chromosome-Scale Scaffolds.

Toll-like receptors (TLRs) are a complex family of innate immune genes that are well characterized in mammals and birds but less well understood in nonavian sauropsids (reptiles). The advent of highly contiguous draft genomes of nonmodel organisms enables study of such gene families through analysis of synteny and sequence identity. Here, we analyze TLR genes from the genomes of 22 tetrapod species. Findings reveal a TLR8 gene expansion in crocodilians and turtles (TLR8B), and a second duplication (TLR8C) specifically within turtles, followed by pseudogenization of that gene in the nonfreshwater species (desert tortoise and green sea turtle). Additionally, the Mojave desert tortoise (Gopherus agassizii) has a stop codon in TLR8B (TLR8-1) that is polymorphic among conspecifics. Revised orthology further reveals a new TLR homolog, TLR21-like, which is exclusive to lizards, snakes, turtles, and crocodilians. These analyses were made possible by a new draft genome assembly of the desert tortoise (gopAga2.0), which used chromatin-based assembly to yield draft chromosomal scaffolds (L50 = 26 scaffolds, N50 = 28.36 Mb, longest scaffold = 107 Mb) and an enhanced de novo genome annotation with 25,469 genes. Our three-step approach to orthology curation and comparative analysis of TLR genes shows what new insights are possible using genome assemblies with chromosome-scale scaffolds that permit integration of synteny conservation data.

A Conserved Kinase-Based Body-Temperature Sensor Globally Controls Alternative Splicing and Gene Expression.

Homeothermic organisms maintain their core body temperature in a narrow, tightly controlled range. Whether and how subtle circadian oscillations or disease-associated changes in core body temperature are sensed and integrated in gene expression programs remain elusive. Furthermore, a thermo-sensor capable of sensing the small temperature differentials leading to temperature-dependent sex determination (TSD) in poikilothermic reptiles has not been identified. Here, we show that the activity of CDC-like kinases (CLKs) is highly responsive to physiological temperature changes, which is conferred by structural rearrangements within the kinase activation segment. Lower body temperature activates CLKs resulting in strongly increased phosphorylation of SR proteins in vitro and in vivo. This globally controls temperature-dependent alternative splicing and gene expression, with wide implications in circadian, tissue-specific, and disease-associated settings. This temperature sensor is conserved across evolution and adapted to growth temperatures of diverse poikilotherms. The dynamic temperature range of reptilian CLK homologs suggests a role in TSD.

Developmental mechanisms driving complex tooth shape in reptiles.

BACKGROUND: In mammals, odontogenesis is regulated by transient signaling centers known as enamel knots (EKs), which drive the dental epithelium shaping. However, the developmental mechanisms contributing to formation of complex tooth shape in reptiles are not fully understood. Here, we aim to elucidate whether signaling organizers similar to EKs appear during reptilian odontogenesis and how enamel ridges are formed. RESULTS: Morphological structures resembling the mammalian EK were found during reptile odontogenesis. Similar to mammalian primary EKs, they exhibit the presence of apoptotic cells and no proliferating cells. Moreover, expression of mammalian EK-specific molecules (SHH, FGF4, and ST14) and GLI2-negative cells were found in reptilian EK-like areas. 3D analysis of the nucleus shape revealed distinct rearrangement of the cells associated with enamel groove formation. This process was associated with ultrastructural changes and lipid droplet accumulation in the cells directly above the forming ridge, accompanied by alteration of membranous molecule expression (Na/K-ATPase) and cytoskeletal rearrangement (F-actin). CONCLUSIONS: The final complex shape of reptilian teeth is orchestrated by a combination of changes in cell signaling, cell shape, and cell rearrangement. All these factors contribute to asymmetry in the inner enamel epithelium development, enamel deposition, ultimately leading to the formation of characteristic enamel ridges.

Waking the sleeping dragon: gene expression profiling reveals adaptive strategies of the hibernating reptile Pogona vitticeps.

BACKGROUND: Hibernation is a physiological state exploited by many animals exposed to prolonged adverse environmental conditions associated with winter. Large changes in metabolism and cellular function occur, with many stress response pathways modulated to tolerate physiological challenges that might otherwise be lethal. Many studies have sought to elucidate the molecular mechanisms of mammalian hibernation, but detailed analyses are lacking in reptiles. Here we examine gene expression in the Australian central bearded dragon (Pogona vitticeps) using mRNA-seq and label-free quantitative mass spectrometry in matched brain, heart and skeletal muscle samples from animals at late hibernation, 2 days post-arousal and 2 months post-arousal. RESULTS: We identified differentially expressed genes in all tissues between hibernation and post-arousal time points; with 4264 differentially expressed genes in brain, 5340 differentially expressed genes in heart, and 5587 differentially expressed genes in skeletal muscle. Furthermore, we identified 2482 differentially expressed genes across all tissues. Proteomic analysis identified 743 proteins (58 differentially expressed) in brain, 535 (57 differentially expressed) in heart, and 337 (36 differentially expressed) in skeletal muscle. Tissue-specific analyses revealed enrichment of protective mechanisms in all tissues, including neuroprotective pathways in brain, cardiac hypertrophic processes in heart, and atrophy protective pathways in skeletal muscle. In all tissues stress response pathways were induced during hibernation, as well as evidence for gene expression regulation at transcription, translation and post-translation. CONCLUSIONS: These results reveal critical stress response pathways and protective mechanisms that allow for maintenance of both tissue-specific function, and survival during hibernation in the central bearded dragon. Furthermore, we provide evidence for multiple levels of gene expression regulation during hibernation, particularly enrichment of miRNA-mediated translational repression machinery; a process that would allow for rapid and energy efficient reactivation of translation from mature mRNA molecules at arousal. This study is the first molecular investigation of its kind in a hibernating reptile, and identifies strategies not yet observed in other hibernators to cope stress associated with this remarkable state of metabolic depression.

Molecular structure of sauropsid ß-keratins from tuatara (Sphenodon punctatus).

The birds and reptiles, collectively known as the sauropsids, can be subdivided phylogenetically into the archosaurs (birds, crocodiles), the testudines (turtles), the squamates (lizards, snakes) and the rhynchocephalia (tuatara). The structural framework of the epidermal appendages from the sauropsids, which include feathers, claws and scales, has previously been characterised by electron microscopy, infrared spectroscopy and X-ray diffraction analyses, as well as by studies of the amino acid sequences of the constituent ß-keratin proteins (also referred to as the corneous ß-proteins). An important omission in this work, however, was the lack of sequence and structural data relating to the epidermal appendages of the rhynchocephalia (tuatara), one of the two branches of the lepidosaurs. Considerable effort has gone into sequencing the tuatara genome and while this is not yet complete, there are now sufficient sequence data for conclusions to be drawn on the similarity of the ß-keratins from the tuatara to those of other members of the sauropsids. These results, together with a comparison of the X-ray diffraction pattern of tuatara claw with those from seagull feather and goanna claw, confirm that there is a common structural plan in the ß-keratins of all of the sauropsids, and not just those that comprise the archosaurs (birds and crocodiles), the testudines (turtles) and the squamates (lizards and snakes).

High arsenic and low lead concentrations in fish and reptiles from Taim wetlands, a Ramsar site in southern Brazil.

The pollution caused by heavy metals and metalloids represent an emerging threat to wetlands worldwide. Herein we examined the concentrations of arsenic (As) and lead (Pb) in fish and aquatic/semi-aquatic reptiles from Taim wetlands, a Ramsar site located at the southernmost Brazilian coastal plain. A total of 82 individuals from six fish and three reptile species from varied trophic levels were analysed through furnace graphite atomic absorption spectrophotometry. Mean As concentrations (µg·g-1 dry weight) were markedly high, ranging from 13.06 ±â€¯3.18 to 19.4 ±â€¯4.04 in fish and 3.51 ±â€¯2.36 to 19.00 ±â€¯10.45 in reptiles. Mean Pb concentrations were low, ranging from 0.00067 ±â€¯0.00060 to 0.0040 ±â€¯0.00045 in fishes and 0.00103 ±â€¯0.0011 to 0.0271 ±â€¯0.0353 in reptiles. The highest As mean level was detected in the herbivore-insectivore fish Astyanax aff. fasciatus, a species of low trophic level among the analysed taxa. The highest Pb mean level was found in the broad-snouted caiman Caiman latirostris, the highest trophic level species analysed. The present study warns for the contamination of As especially in edible fish, which constitute a threat to the communities that use this resource in systems connected to Taim wetlands. As concentrations in reptiles were also higher than those reported in previous studies concerning the groups herein addressed. It is possible that the high As burdens found in the analysed species could be attributed to the use of fertilizers and pesticides in extensive irrigated rice areas located in Taim wetlands surroundings, but natural sources cannot be dismissed.

5-Methylcytosine-Rich Heterochromatin in Reptiles.

An experimental approach using monoclonal anti-5-methylcytosine antibodies and indirect immunofluorescence was elaborated for detecting 5-methylcytosine-rich chromosome regions in reptilian chromosomes. This technique was applied to conventionally prepared mitotic metaphases of 2 turtle species and 12 squamate species from 8 families. The hypermethylation patterns were compared with C-banding patterns obtained by conventional banding techniques. The hypermethylated DNA sequences are species-specific and are located in constitutive heterochromatin. They are highly reproducible and often found in centromeric, pericentromeric, and interstitial positions of the chromosomes. Heterochromatic regions in differentiated sex chromosomes are particularly hypermethylated.

FoxP2 Expression in a Highly Vocal Teleost Fish with Comparisons to Tetrapods.

Motivated by studies of speech deficits in humans, several studies over the past two decades have investigated the potential role of a forkhead domain transcription factor, FoxP2, in the central control of acoustic signaling/vocalization among vertebrates. Comparative neuroanatomical studies that mainly include mammalian and avian species have mapped the distribution of FoxP2 expression in multiple brain regions that imply a greater functional significance beyond vocalization that might be shared broadly across vertebrate lineages. To date, reports for teleost fish have been limited in number and scope to nonvocal species. Here, we map the neuroanatomical distribution of FoxP2 mRNA expression in a highly vocal teleost, the plainfin midshipman (Porichthys notatus). We report an extensive overlap between FoxP2 expression and vocal, auditory, and steroid-signaling systems with robust expression at multiple sites in the telencephalon, the preoptic area, the diencephalon, and the midbrain. Label was far more restricted in the hindbrain though robust in one region of the reticular formation. A comparison with other teleosts and tetrapods suggests an evolutionarily conserved FoxP2 phenotype important to vocal-acoustic and, more broadly, sensorimotor function among vertebrates.

Review: Evolution and diversification of corneous beta-proteins, the characteristic epidermal proteins of reptiles and birds.

In all amniotes specialized intermediate filament keratins (IF-keratins), in addition to keratin-associated and corneous proteins form the outermost cornified layer of the epidermis. Only in reptiles and birds (sauropsids) the epidermis of scales, claws, beaks, and feathers, largely comprises small proteins formerly indicated as "beta-keratins" but here identified as corneous beta-proteins (CBPs) to avoid confusion with true keratins. Genes coding for CBPs have evolved within the epidermal differentiation complex (EDC), a locus with no relationship with those of IF-keratins. CBP genes have the same exon-intron structure as EDC genes encoding other corneous proteins of sauropsids and mammals, but they are unique by encoding a peculiar internal amino acid sequence motif beta-sheet region that allows formation of CBP filaments in the epidermis and epidermal appendages of reptiles and birds. In contrast, skin appendages of mammals, like hairs, claws, horns and nails, contain keratin-associated proteins that, like IF-keratin genes, are encoded by genes in loci different from the EDC. Phylogenetic analysis shows that lepidosaurian (lizards and snakes) and nonlepidosaurian (crocodilians, birds, and turtles) CBPs form two separate clades that likely originated after the divergence of these groups of sauropsids in the Permian Period. Clade-specific CBPs evolved to make most of the corneous material of feathers in birds and of the shell in turtles. Based on the recent identification of the complete sets of CBPs in all major phylogenetic clades of sauropsids, this review provides a comprehensive overview of the molecular evolution of CBPs.

Effects of cathelicidin-derived peptide from reptiles on lipopolysaccharide-induced intestinal inflammation in weaned piglets.

Cathelicidins are the largest family of antimicrobial peptides. C-BF, which is short for Cathelicidin-Bungarus Fasciatus, was isolated from snake venom. C-BF was found to be the most potential substitutes for antibiotics. In this study, we analyzed the effects of cathelicidin-derived peptide C-BF, on lipopolysaccharide (LPS)-induced intestinal damage in weaned piglets, to evaluate the therapeutic effect of C-BF on infectious disease of piglets. Twenty-four piglets were randomly assigned into four groups: control, C-BF, LPS, and C-BF+LPS. The LPS and C-BF+LPS groups were intraperitoneally injected with LPS at fixed timepoints, while the control and C-BF groups were injected with equal volumes of saline. The C-BF and C-BF+LPS groups were then intraperitoneally injected with antimicrobial peptide C-BF, while the control and LPS groups were injected with equal volumes of saline. All piglets were observed for 15days and then sacrificed for analysis. The results showed that C-BF significantly improved the growth performance of weaned piglets compared with LPS-treated animals (P<0.05), and that C-BF could ameliorate the structural and developmental damage to the small intestine caused by LPS treatment. Further, the level of apoptosis in the LPS group was significantly higher than in the other three groups (P<0.05), as was the invasion of inflammatory cells into the intestinal mucosa of the jejunum (P<0.05), leading to increased secretion of pro-inflammatory cytokines. In conclusion, the study indicates that C-BF treatment may be a potential therapy for LPS/pathogen-induced intestinal injury in piglets.

Morpho-functional characterization of the systemic venous pole of the reptile heart.

Mammals evolved from reptile-like ancestors, and while the mammalian heart is driven by a distinct sinus node, a sinus node is not apparent in reptiles. We characterized the myocardial systemic venous pole, the sinus venosus, in reptiles to identify the dominant pacemaker and to assess whether the sinus venosus remodels and adopts an atrium-like phenotype as observed in mammals. Anolis lizards had an extensive sinus venosus of myocardium expressing Tbx18. A small sub-population of cells encircling the sinuatrial junction expressed Isl1, Bmp2, Tbx3, and Hcn4, homologues of genes marking the mammalian sinus node. Electrical mapping showed that hearts of Anolis lizards and Python snakes were driven from the sinuatrial junction. The electrical impulse was delayed between the sinus venosus and the right atrium, allowing the sinus venosus to contract and aid right atrial filling. In proximity of the systemic veins, the Anolis sinus venosus expressed markers of the atrial phenotype Nkx2-5 and Gja5. In conclusion, the reptile heart is driven by a pacemaker region with an expression signature similar to that of the immature sinus node of mammals. Unlike mammals, reptiles maintain a sinuatrial delay of the impulse, allowing the partly atrialized sinus venosus to function as a chamber.