Evolution Of Reptiles Research Articles

Trifold origin of the reptilian ear ossicle and its relation to the evolutionary modification of the temporal skull region.

Whereas mammals are characterized by the presence of three middle ear ossicles, reptiles have only one, the columella (stapes). Nevertheless, there is a great diversity of columellar anatomy among sauropsids, especially in the unique and cartilaginous "extracolumella"-portion. Molecular studies revealed the "columella" of chicken and quails to be formed within the second pharyngeal arch, although conflicting evidence exists for the columellar footplate and distal parts of the columella in these birds. We studied columellar development in four turtles, one lizard, and one caiman species and argue, using early blastematous stages, that, distally, the so-called "extracolumella" in turtles is mainly of quadrate, that is, first pharyngeal arch origin. Differently, the dorsal aspect of the "extracolumella" of the lizard and a part of the "dorsal columella process" of the caiman are likely quadrate-derived. This indicates only a partial homology of the distal columellar compartments among reptiles. Moreover, we observed in most species that, at early stages, the footplate differentiates from the otic capsule, which confirms widespread experimental findings of mesodermal cells contributing to the proximal part of the columella. We provide a hypothetical framework for the changes in the columella and quadrate morphology in reptilian evolution. Originally, as evidenced by the fossil record, the columella served as a stabilizing brace between the quadrate and braincase. Associated with changes in the feeding mode of late Permian taxa, the quadrate was integrated along the stress flows from biting, and in early development part of the quadrate differentiated to differently contribute to the distal part of the "columella-complex," which now contacts the tympanic membrane. In addition, part of the original otic capsule contributes to the footplate of the mobile columella, providing a connection with the inner ear.

Chamaeleo calyptratus (veiled chameleon) chromosome-scale genome assembly and annotation provides insights into the evolution of reptiles and developmental mechanisms.

The family Chamaeleonidae comprises 228 species, boasting an extensive geographic spread and an array of evolutionary novelties and adaptations, but a paucity of genetic and molecular analyses. Veiled chameleon (Chamaeleo calyptratus) has emerged as a tractable research organism for the study of squamate early development and evolution. Here we report a chromosomal-level assembly and annotation of the veiled chameleon genome. We note a remarkable chromosomal conservation across squamates, but comparisons to more distant genomes reveal GC peaks correlating with ancestral chromosome fusion events. We subsequently identified the XX/XY region on chromosome 5, confirming environmental-independent sex determination in veiled chameleons. Furthermore, our analysis of the Hox gene family indicates that veiled chameleons possess the most complete set of 41 Hox genes, retained from an amniote ancestor. Lastly, the veiled chameleon genome has retained both ancestral paralogs of the Nodal gene, but is missing Dand5 and several other genes, recently associated with the loss of motile cilia during the establishment of left-right patterning. Thus, a complete veiled chameleon genome provides opportunities for novel insights into the evolution of reptilian genomes and the molecular mechanisms driving phenotypic variation and ecological adaptation.

Developmental energetics in the oviparous corn snake, Pantherophis guttatus, confirms a conservative evolutionary pattern in snakes

Eggs of oviparous reptiles are ideal models for studying evolutionary patterns of embryonic metabolism since they allow tracking of energy allocation during development. Analyzing oxygen consumption of whole eggs throughout development indicates three patterns among reptiles. Embryos initially grow and consume oxygen exponentially, but oxygen consumption slows, or drops before hatching in some species. Turtles, crocodilians, and most lizards follow curves with initial exponential increases followed by declines, whereas embryonic snakes that have been studied exhibit a consistently exponential pattern. This study measured oxygen consumption of corn snake, Pantherophis guttatus, embryos to determine if this species also exhibits an exponential increase in oxygen consumption. Individual eggs, sampled weekly from oviposition to hatching, were placed in respirometry chambers for 24-h during which oxygen consumption was recorded. Embryos were staged and carcasses and yolk were weighed separately. Results indicate steady inclines in oxygen consumption during early stages of development, with a rapid increase prior to hatching. The findings support the hypothesis that embryonic oxygen consumption of snakes differs from most other non-avian reptiles. Total energy required for development was determined based on calorimetry of initial yolk compared to hatchlings and residual yolk and by integration of the area under the curve plotting oxygen consumption versus age of embryos. The cost of development estimates based on these two methods were 6.4 and 10.0 kJ, respectively. Our results emphasize the unique physiological aspects of snake embryogenesis and illustrate how the study of physiological characteristics can contribute to the broader understanding of reptilian evolution.

Филогенетика и географическое распространение скальных ящериц (Lacertidae, Reptilia) на горной системе Эльбурс

Rock lizards of the genus Darevskia have long been an important model object for study of reptile evolution. To understand the overall picture, it is important to know how bisexual and parthenogenetic species are distributed within this genus. The ranges of Caucasian species of rock lizards have been studied for a long time and in detail. However, recent attention to the species inhabiting the territory of Iran has been attracted after the description of several new species in 2013. As part of the continuation of these studies, we studied the distribution and genetic diversity of five species of lizards of the Darevskia genus along the Alborz mountain range in Iran: D. chlorogaster, D. caspica, D. defilippii, D. schaekeli and D. raddei. In the course of this, we discovered new localities of D. defilippii that are outside their known range. We assessed the phylogenetic relationships between the studied species according to the ND4 – Leu tRNA region of mitochondrial DNA and found a relatively high level of genetic variability in D. defilippii and D. chlorogaster, while D. raddei, despite its wide distribution, has low variability. In general, the phylogenetic position of the studied species is somewhat different from that described by Ahmadzadeh et al. (2013).

Successive climate crises in the deep past drove the early evolution and radiation of reptiles.

Climate change–induced mass extinctions provide unique opportunities to explore the impacts of global environmental disturbances on organismal evolution. However, their influence on terrestrial ecosystems remains poorly understood. Here, we provide a new time tree for the early evolution of reptiles and their closest relatives to reconstruct how the Permian-Triassic climatic crises shaped their long-term evolutionary trajectory. By combining rates of phenotypic evolution, mode of selection, body size, and global temperature data, we reveal an intimate association between reptile evolutionary dynamics and climate change in the deep past. We show that the origin and phenotypic radiation of reptiles was not solely driven by ecological opportunity following the end-Permian extinction as previously thought but also the result of multiple adaptive responses to climatic shifts spanning 57 million years.

Early Origins of Divergent Patterns of Morphological Evolution on the Mammal and Reptile Stem-Lineages.

The origin of amniotes 320 million years ago signaled independence from water in vertebrates and was closely followed by divergences within the mammal and reptile stem lineages (Synapsida and Reptilia). Early members of both groups had highly similar morphologies, being superficially “lizard-like” forms with many plesiomorphies. However, the extent to which they might have exhibited divergent patterns of evolutionary change, with the potential to explain the large biological differences between their living members, is unresolved. We use a new, comprehensive phylogenetic dataset to quantify variation in rates and constraints of morphological evolution among Carboniferous–early Permian amniotes. We find evidence for an early burst of evolutionary rates, resulting in the early origins of morphologically distinctive subgroups that mostly persisted through the Cisuralian. Rates declined substantially through time, especially in reptiles. Early reptile evolution was also more constrained compared with early synapsids, exploring a more limited character state space. Postcranial innovation in particular was important in early synapsids, potentially related to their early origins of large body size. In contrast, early reptiles predominantly varied the temporal region, suggesting disparity in skull and jaw kinematics, and foreshadowing the variability of cranial biomechanics seen in reptiles today. Our results demonstrate that synapsids and reptiles underwent an early divergence of macroevolutionary patterns. This laid the foundation for subsequent evolutionary events and may be critical in understanding the substantial differences between mammals and reptiles today. Potential explanations include an early divergence of developmental processes or of ecological factors, warranting cross-disciplinary investigation. [Amniote; body size; constraint; phylogeny; rate.]

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.

De novo Assembly, Annotation, and Analysis of Transcriptome Data of the Ladakh Ground Skink Provide Genetic Information on High-Altitude Adaptation

The Himalayan Arc is recognized as a global biodiversity hotspot. Among its numerous cryptic and undiscovered organisms, this composite high-mountain ecosystem harbors many taxa with adaptations to life in high elevations. However, evolutionary patterns and genomic features have been relatively rarely studied in Himalayan vertebrates. Here, we provide the first well-annotated transcriptome of a Greater Himalayan reptile species, the Ladakh Ground skink Asymblepharus ladacensis (Squamata: Scincidae). Based on tissues from the brain, an embryonic disc, and pooled organ material, using pair-end Illumina NextSeq 500 RNAseq, we assembled ~77,000 transcripts, which were annotated using seven functional databases. We tested ~1600 genes, known to be under positive selection in anurans and reptiles adapted to high elevations, and potentially detected positive selection for 114 of these genes in Asymblepharus. Even though the strength of these results is limited due to the single-animal approach, our transcriptome resource may be valuable data for further studies on squamate reptile evolution in the Himalayas as a hotspot of biodiversity.

Whole-chromosome fusions in the karyotype evolution ofSceloporus(Iguania, Reptilia) are more frequent in sex chromosomes than autosomes

Whole-chromosome fusions play a major role in the karyotypic evolution of reptiles. It has been suggested that certain chromosomes tend to fuse with sex chromosomes more frequently than others. However, the comparative genomic synteny data are too scarce to draw strong conclusions. We obtained and sequenced chromosome-specific DNA pools of Sceloporus malachiticus, an iguanian species which has experienced many chromosome fusions. We found that four of seven lineage-specific fusions involved sex chromosomes, and that certain syntenic blocks which constitute the sex chromosomes, such as the homologues of the Anolis carolinensis chromosomes 11 and 16, are repeatedly involved in sex chromosome formation in different squamate species. To test the hypothesis that the karyotypic shift could be associated with changes in recombination patterns, we performed a synaptonemal complex analysis in this species and in Sceloporus variabilis (2n = 34). It revealed that the sex chromosomes in S. malachiticus had two distal pseudoautosomal regions and a medial differentiated region. We found that multiple fusions little affected the recombination rate in S. malachiticus. Our data confirm more frequent involvement of certain chromosomes in sex chromosome formation, but do not reveal a connection between the gonosome-autosome fusions and the evolution of recombination rate. This article is part of the theme issue 'Challenging the paradigm in sex chromosome evolution: empirical and theoretical insights with a focus on vertebrates (Part II)'.

Tracking the Origin and Early Evolution of Reptiles

The origin of Reptilia and the biostratigraphic and palaeobiogeographic distribution of its early representatives are still poorly understood. An independent source of information may come from the extensive Carboniferous footprint record of reptiles, which is arguably richer and more complete than the skeletal record. Nevertheless, previous studies often failed to provide useful information because they were based on poorly preserved material and/or characters non-exclusive of reptile tracks. In fact, a large part of the supposed early reptile tracks can be assigned to the anamniote ichnotaxon Hylopus hardingi. Here, we revise the ichnotaxon Hylopus hardingi based on anatomy-consistent material, attribute it to anamniote reptiliomorphs, and distinguish it from Notalacerta missouriensis, the earliest ichnotaxon that can be attributed to reptiles, and the somewhat younger Varanopus microdactylus (attributed to parareptiles, such as bolosaurians) and Dromopus lacertoides (attributed to araeoscelid reptiles and non-varanodontine varanopids). These attributions are based on correlating morphofunctional features of tracks and skeletons. Multivariate analysis of trackway parameters indicates that the late Bashkirian Notalacerta missouriensis and Hylopus hardingi differ markedly in their trackway patterns from Late Mississippian Hylopus hardingi and Late Pennsylvanian reptile tracks, which appear to share a derived amniote-like type of gait. While the first occurrence/appearance of reptile tracks in the tetrapod footprint record during the late Bashkirian corresponds to the first occurrence/appearance of reptiles in the skeletal record, footprints significantly enlarge the paleobiogeographic distribution of the group, suggesting an earlier radiation of reptiles during the Bashkirian throughout North America and possibly North Africa. Dromopus appeared in the Kasimovian together with the diapsid group Araeoscelidia, but footprints from Western-European occurrences enlarge the paleobiogeographic distribution of diapsids and varanopids. Varanopus and bolosaurian parareptiles appear in the Gzhelian of North America. Older parareptiles are, however, known from the late Moscovian. In all, the footprint record of early reptiles supplements the skeletal record, suggesting possible future lines of research.

Independent pseudogenizations and losses of sox15 during amniote diversification following asymmetric ohnolog evolution

BackgroundFour ohnologous genes (sox1, sox2, sox3, and sox15) were generated by two rounds of whole-genome duplication in a vertebrate ancestor. In eutherian mammals, Sox1, Sox2, and Sox3 participate in central nervous system (CNS) development. Sox15 has a function in skeletal muscle regeneration and has little functional overlap with the other three ohnologs. In contrast, the frog Xenopus laevis and zebrafish orthologs of sox15 as well as sox1-3 function in CNS development. We previously reported that Sox15 is involved in mouse placental development as neofunctionalization, but is pseudogenized in the marsupial opossum. These findings suggest that sox15 might have evolved with divergent gene fates during vertebrate evolution. However, knowledge concerning sox15 in other vertebrate lineages than therian mammals, anuran amphibians, and teleost fish is scarce. Our purpose in this study was to clarify the fate and molecular evolution of sox15 during vertebrate evolution.ResultsWe searched for sox15 orthologs in all vertebrate classes from agnathans to mammals by significant sequence similarity and synteny analyses using vertebrate genome databases. Interestingly, sox15 was independently pseudogenized at least twice during diversification of the marsupial mammals. Moreover, we observed independent gene loss of sox15 at least twice during reptile evolution in squamates and crocodile-bird diversification. Codon-based phylogenetic tree and selective analyses revealed an increased dN/dS ratio for sox15 compared to the other three ohnologs during jawed vertebrate evolution.ConclusionsThe findings revealed an asymmetric evolution of sox15 among the four ohnologs during vertebrate evolution, which was supported by the increased dN/dS values in cartilaginous fishes, anuran amphibians, and amniotes. The increased dN/dS value of sox15 may have been caused mainly by relaxed selection. Notably, independent pseudogenizations and losses of sox15 were observed during marsupial and reptile evolution, respectively. Both might have been caused by strong relaxed selection. The drastic gene fates of sox15, including neofunctionalization and pseudogenizations/losses during amniote diversification, might be caused by a release from evolutionary constraints.

Genomic evidence of adaptive evolution in the reptilian SOCS gene family.

The suppressor of the cytokine signaling (SOCS) family of proteins play an essential role in inhibiting cytokine receptor signaling by regulating immune signal pathways. Although SOCS gene functions have been examined extensively, no comprehensive study has been performed on this gene family’s molecular evolution in reptiles. In this study, we identified eight canonical SOCS genes using recently-published reptilian genomes. We used phylogenetic analysis to determine that the SOCS genes had highly conserved evolutionary dynamics that we classified into two types. We identified positive SOCS4 selection signals in whole reptile lineages and SOCS2 selection signals in the crocodilian lineage. Selective pressure analyses using the branch model and Z-test revealed that these genes were under different negative selection pressures compared to reptile lineages. We also concluded that the nature of selection pressure varies across different reptile lineages on SOCS3, and the crocodilian lineage has experienced rapid evolution. Our results may provide a theoretical foundation for further analyses of reptilian SOCS genes’ functional and molecular mechanisms, as well as their roles in reptile growth and development.

Revisiting the Karyotypes of Alligators and Caimans (Crocodylia, Alligatoridae) after a Half-Century Delay: Bridging the Gap in the Chromosomal Evolution of Reptiles.

Although crocodilians have attracted enormous attention in other research fields, from the cytogenetic point of view, this group remains understudied. Here, we analyzed the karyotypes of eight species formally described from the Alligatoridae family using differential staining, fluorescence in situ hybridization with rDNA and repetitive motifs as a probe, whole chromosome painting (WCP), and comparative genome hybridization. All Caimaninae species have a diploid chromosome number (2n) 42 and karyotypes dominated by acrocentric chromosomes, in contrast to both species of Alligatorinae, which have 2n = 32 and karyotypes that are predominantly metacentric, suggesting fusion/fission rearrangements. Our WCP results supported this scenario by revealing the homeology of the largest metacentric pair present in both Alligator spp. with two smaller pairs of acrocentrics in Caimaninae species. The clusters of 18S rDNA were found on one chromosome pair in all species, except for Paleosuchus spp., which possessed three chromosome pairs bearing these sites. Similarly, comparative genomic hybridization demonstrated an advanced stage of sequence divergence among the caiman genomes, with Paleosuchus standing out as the most divergent. Thus, although Alligatoridae exhibited rather low species diversity and some level of karyotype stasis, their genomic content indicates that they are not as conserved as previously thought. These new data deepen the discussion of cytotaxonomy in this family.

Development of the embryonic liver and pancreas of the Chinese softshell turtle Trionyx sinensis

ABSTRACT The research on hatching ecology of the Chinese softshell turtle Trionyx sinensis has essential guiding roles to clarify the physiological and ecological mechanism of reptile evolution. The aim of this study is to describe the histological changes, differentiation, and maturation of some functional cells during the genesis and development of the liver and pancreas of the Chinese softshell turtle T. sinensis. Softshell turtle eggs were incubated under artificial conditions and hatched within 41–45 days. Hematoxylin and eosin-stained embryonic pancreas and liver were examined at various time points from 2 to 31 days and compared with that of other reptiles, amphibians, fishes, and birds in the literature. Immunohistochemical assay for glucagon and insulin was performed on paraformaldehyde-fixed embryos to identify functional cells in the pancreas. Pancreatic endocrine cells of T. sinensis have secretory ability at day 26 of embryonic development, and the dispersed pancreatic endocrine cells may be the result of the incomplete pancreatic development.

Integrative Comparative Cognition: Can Neurobiology and Neurogenomics Inform Comparative Analyses of Cognitive Phenotype?

A long-standing question in animal behavior is what are the patterns and processes that shape the evolution of cognition? One effective way to address this question is to study cognitive abilities in a broad spectrum of animals. While comparative psychologists have traditionally focused on a narrow range of organisms, today they may work with any number of species, from frogs to birds or bees. This broader range of study species has greatly enriched our understanding of the diversity of cognitive processes among animals. Yet, this diversity has highlighted the fundamental challenge of comparing cognitive processes across animals. An analysis of the neural and molecular mechanisms of cognition may be necessary to solve this problem. The goal of our symposium was to bring together speakers studying a range of species to gain a broadly integrative perspective on cognition while at the same time considering the potentially important role of neurobiology and genomics in addressing the difficult problem of comparing cognition across species. For example, work by MaBouDi et al. indicates that neural constraints on computing power may impact the cognitive processes underlying numerical discrimination in bees. A presentation by Lara LaDage demonstrated how neurobiology can be used to better understand cognition and its evolution in reptiles while Edwards et al. identify the cerebellum as potentially important in the performance of the complex process of nest building. We see that molecular approaches highlight the contributions of the prefrontal cortex and hippocampus to cognitive phenotype across vertebrates while, at the same time, identifying the genes and cellular processes that may contribute to evolution of cognition. The potentially important role of neurogenesis and synaptic plasticity emerge clearly from such studies. Still unanswered is the question of whether molecular tools will contribute to our ability to discriminate convergent/parallel evolution from homology in the evolution of cognitive phenotype.

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.

Conserved visual sensitivities across divergent lizard lineages that differ in an ultraviolet sexual signal.

The sensory drive hypothesis predicts the correlated evolution of signaling traits and sensory perception in differing environments. For visual signals, adaptive divergence in both color signals and visual sensitivities between populations may contribute to reproductive isolation and promote speciation, but this has rarely been tested or shown in terrestrial species. We tested whether opsin protein expression differs between divergent lineages of the tawny dragon (Ctenophorus decresii) that differ in the presence/absence of an ultraviolet sexual signal. We measured the expression of four retinal cone opsin genes (SWS1, SWS2, RH2, and LWS) using droplet digital PCR. We show that gene expression between lineages does not differ significantly, including the UV wavelength sensitive SWS1. We discuss these results in the context of mounting evidence that visual sensitivities are highly conserved in terrestrial systems. Multiple competing requirements may constrain divergence of visual sensitivities in response to sexual signals. Instead, signal contrast could be increased via alternative mechanisms, such as background selection. Our results contribute to a growing understanding of the roles of visual ecology, phylogeny, and behavior on visual system evolution in reptiles.

CRISPR-Cas9 Gene Editing in Lizards through Microinjection of Unfertilized Oocytes

CRISPR-Cas9-mediated gene editing has enabled the direct manipulation of gene function in many species. However, the reproductive biology of reptiles presents unique barriers for the use of this technology, and there are no reptiles with effective methods for targeted mutagenesis. Here, we demonstrate thatthe microinjection of immature oocytes within theovaries of Anolis sagrei females enables the production of CRISPR-Cas9-induced mutations. This method is capable of producing F0 embryos and hatchlings with monoallelic or biallelic mutations. We demonstrate that these mutations can be transmitted through the germline to establish genetically modified strains of lizards. Direct tests of gene function can now be performed in Anolis lizards, an important model for studies of reptile evolution and development.

Genomic analysis and adaptive evolution of the RIG-I-like and NOD-like receptors in reptiles

Pattern recognition receptors (PRRs) play a crucial role in the host's innate immune system. Among the PPRs, the RIG-I-like (RLRs) and NOD-like receptors (NLRs) are two important subgroups. To understand the role of RLRs (RIG-I, MDA5, LGP2) and NLRs (NOD1, NLRC3, NLRX1, NOD2) in reptilian evolution, we identified six genes from reptilian genomes. A total of 168 putative genes were identified from 28 reptile species, including 141 intact genes, 25 partial genes and two pseudogenes. Interestingly, the NOD2 gene was absent in all reptile species. Phylogenetic results showed that all genes were divided into four major clades corresponding to their traditional taxonomic groups, indicating that these genes are conserved in reptiles. Evolutionary analyses detected positive selection in six genes. Most of the positively selected sites (50/76) were located in known functional domains, reflecting their critical and particular contributions to host defense during reptilian evolution. Branch model analysis showed that NLRs were under different evolutionary forces, while the RLRs were not, suggesting that semiaquatic species and terrestrial species faced different environmental pathogens, leading to different adaptations. Moreover, the positively selected sites identified in MDA5 using the branch-site model among semiaquatic reptiles, suggested their involvement in adaptation to semiaquatic environments.

Squamate Chromosome Size and GC Content Assessed by Flow Karyotyping

Chromosome homologies in reptiles have been investigated extensively by gene mapping and chromosome painting. Relative chromosome size can be estimated roughly from conventional karyotypes, but chromosome GC content cannot be evaluated by any of these approaches. However, GC content can be obtained by whole-genome sequencing, although complete data are available only for a limited number of reptilian species. Chromosomes can be characterized by size and GC content in bivariate flow karyotypes, in which the distribution of peaks represents the differences. We have analysed flow karyotypes from 9 representative squamate species and show chromosome profiles for each species based on the relationship between size and GC content. Our results reveal that the GC content of macrochromosomes is invariable in the 9 species. A higher GC content was found in microchromosomes, similar to profiles previously determined in crocodile, turtle, and chicken. The findings suggest that karyotype evolution in reptiles is characterized by unique features of chromosome GC content.