Integrative and Comparative Biology Advance Access originally published online on June 24, 2008
Integrative and Comparative Biology 2008 48(4):454-462; doi:10.1093/icb/icn062
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Turtle isochore structure is intermediate between amphibians and other amniotes
Department of Zoology, University of Florida, 223 Bartram Hall, PO Box 118525, Gainesville, FL 32611, USA
Correspondence: 1E-mail: jena{at}zoo.ufl.edu
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Vertebrate genomes are comprised of isochores that are relatively long (>100 kb) regions with a relatively homogenous (either GC-rich or AT-rich) base composition and with rather sharp boundaries with neighboring isochores. Mammals and living archosaurs (birds and crocodilians) have heterogeneous genomes that include very GC-rich isochores. In sharp contrast, the genomes of amphibians and fishes are more homogeneous and they have a lower overall GC content. Because DNA with higher GC content is more thermostable, the elevated GC content of mammalian and archosaurian DNA has been hypothesized to be an adaptation to higher body temperatures. This hypothesis can be tested by examining structure of isochores across the reptilian clade, which includes the archosaurs, testudines (turtles), and lepidosaurs (lizards and snakes), because reptiles exhibit diverse body sizes, metabolic rates, and patterns of thermoregulation. This study focuses on a comparative analysis of a new set of expressed genes of the red-eared slider turtle and orthologs of the turtle genes in mammalian (human, mouse, dog, and opossum), archosaurian (chicken and alligator), and amphibian (western clawed frog) genomes. EST (expressed sequence tag) data from a turtle cDNA library enriched for genes that have specialized functions (developmental genes) revealed using the GC content of the third-codon-position to examine isochore structure requires careful consideration of the types of genes examined. The more highly expressed genes (e.g., housekeeping genes) are more likely to be GC-rich than are genes with specialized functions. However, the set of highly expressed turtle genes demonstrated that the turtle genome has a GC content that is intermediate between the GC-poor amphibians and the GC-rich mammals and archosaurs. There was a strong correlation between the GC content of all turtle genes and the GC content of other vertebrate genes, with the slope of the line describing this relationship also indicating that the isochore structure of turtles is intermediate between that of amphibians and other amniotes. These data are consistent with some thermal hypotheses of isochore evolution, but we believe that the credible set of models for isochore evolution still includes a variety of models. These data expand the amount of genomic data available from reptiles upon which future studies of reptilian genomics can build.
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Introduction |
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Vertebrate genomes are mosaics of isochores, defined as long (>100 kb) regions of relatively homogenous base composition (either GC-rich or AT-rich) that have sharp boundaries with neighboring regions (Bernardi 2000
). One motivation for the study of isochore evolution is the observation that "warm-blooded" (hereafter homeothermic) mammals and birds exhibit substantial differences from "cold-blooded" (hereafter poikilothermic) amphibians and fish in heterogeneity of their genomic GC content (Bernardi 2000). Mammals and birds have isochores with as much as 60% GC and other genomic regions with as little as 30% GC, while amphibians and fish have more homogenous and often less GC-rich genomes. The best-characterized isochore structures include the human (Costantini et al. 2006), mouse (Zhang and Zhang 2004), and chicken (Gao and Zhang 2006; Costantini et al. 2007a, 2007b) genomes, while amphibian (Fortes et al. 2007), crocodilian (Chojnowski et al. 2007), fish (Costantini et al. 2007a), and marsupial (Gu et al. 2007) genomes are somewhat less well-characterized. Many fundamental biological properties (e.g., gene density, recombination) are correlated with isochore structure (reviewed by Bernardi 2000; Eyre-Walker and Hurst 2001). In fact, the map of the human isochore revealed that the number and distribution of isochores is consistent with the patterns of chromosome banding during prophase (Costantini et al. 2006), emphasizing that isochores are fundamental to vertebrate genomic organization and evolution.
Models of isochore evolution have invoked either mutational biases (leading to patterns of neutral evolution that generate isochores) or natural selection (Bernardi 2007; Eyre-Walker and Hurst 2001). However, a second major division exists between models with a thermal basis and those that are nonthermal (Chojnowski et al. 2007). The most complete thermal hypotheses are also based upon selection, and they suggest that the evolution of GC-rich isochores is largely based on the greater stability of GC-rich regions at high temperature (Bernardi 2000, 2007). However, there may also be selection for specific types of protein encoded by genes in GC-rich isochores, since those proteins differ structurally from proteins encoded by genes in GC-poor isochores (Chiusano et al. 1999; D’Onofrio et al. 1999). This can be called the genomic stability hypothesis as it is largely based upon the notion that GC-rich isochores are an adaptation to constantly high body temperatures. The deamination feedback loop (Fryxell and Zuckerkandl 2000) is a neutral (mutational) thermal hypothesis, since it postulates that GC-poor regions have a higher rate of cytosine deamination (which results in C
T transitions) at high temperatures, further reducing their GC content. However, the deamination feedback loop predicts a unidirectional bias that explains homeothermic isochore structures only if the ancestral condition was GC-rich, which is not suggested by the relatively homogeneous GC-poor genomes in amphibians and fishes. The observation that the poikilothermic American alligator has a GC-rich isochore structure similar to that of mammals and birds (Chojnowski et al. 2007), adds additional emphasis that GC-rich isochores cannot be an adaptation to homeothermy per se. However, Chojnowski et al. (2007) pointed out that some poikilotherms maintain a body temperature similar to that of many homeotherms for relatively long periods (Seebacher and Shine 2004), and suggested that thermal models of isochore evolution should consider this fact.
A model unrelated to thermal factors and based upon natural selection is the "epigenetic optimization hypothesis" of Vinogradov (2005), which postulates that base composition is optimized for active transcription of GC-rich regions and suppression of GC-poor regions. It is unclear, however, how the epigenetic optimization hypothesis explains the transition from the more homogeneous genomes of amphibians and fish to the more heterogeneous mammalian and archosaurian genomes. One nonthermal mutational hypothesis invokes distinct biases for regions with different replication times; early and late replicating sequences are proposed to exhibit different patterns of mutation that reflect changes in the free nucleotide pools (Wolfe et al. 1989). Although one study reported that isochore structure and replication time are uncorrelated in somatic mammalian cells (Eyre-Walker 1992), replication times can change during differentiation (Hiratani et al. 2004), and other studies have found a good correlation (Schmegner et al. 2007). Different patterns of DNA repair might also result in changes to the overall base composition (Boulikas 1992), although it is unclear whether these biases extend over the length of typical isochore structure. However, the mutational model invoked most commonly is biased gene conversion (Duret et al. 2006; Li et al. 2007), a process linked to recombination that results in a higher probability of GC alleles converting AT alleles than is true of the reverse.
Despite their potentially critical role for understanding isochore evolution, there is a surprising lack of information about reptilian isochore structure. There are three major lineages of living reptiles: lepidosaurs (lizards, snakes, and tuatara), testudines (turtles), and archosaurs (crocodilians and birds) (Rest et al. 2003; Iwabe et al. 2005; Hugall et al. 2007). This emphasizes that it is inappropriate to consider reptiles homogeneous. In fact, reptiles exhibit diverse body sizes, metabolic rates, and patterns of thermoregulation (Zug et al. 2001), so it is important to learn more about reptilian isochore structures to examine the relationship between GC content and thermal biology. The large-scale study by Chojnowski et al. (2007) extended and strongly supported earlier studies based upon a few genes (Hughes et al. 1999) by demonstrating that crocodilians and birds have similar isochore structures. Thus, the GC-rich isochore structure of birds is actually an archosaurian phenomenon. Archosaurs, in turn, have an isochore structure similar to that of mammals. It remains unclear, however, whether GC-rich isochore structure is a feature of amniote genomes or the product of convergent evolution between archosaurs and mammals. To define the isochore structure of another reptilian lineage, we examined the GC content of multiple expressed genes in the red-eared slider turtle (Trachemys scripta) and compared this to results from organisms with known isochore structures.
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Collection and assembly of ESTs
Three subtraction libraries of turtles were generated using the suppression subtraction hybridization method (Diatchenko et al. 1996
) with the PCR-SelectTM cDNA Subtraction Kit (Clontech, Mountain View, CA). The source of the mRNAs used to generate the libraries was whole turtle embryos that were incubated at different temperatures or with estradiol (manuscript in preparation; for additional details regarding the library, contact J.L.C.). The use of suppression subtraction hybridization is expected to enrich the library for conditionally expressed transcripts relative to housekeeping genes. A total of 1983 expressed sequence tags (ESTs; deposited in dbEST with accession numbers FG341000
[GenBank]
to FG341832
[GenBank]
), were obtained from the turtle subtraction cDNA libraries using an Applied Biosystems (ABI 3100 Avant, Foster City, CA USA) automated sequencer. ESTs were assembled as described by Liang et al. (2000).
EST assemblies were used as tBLASTx (Altschul et al. 1997) queries to search cDNA sequences from the EnsEMBL database (Birney et al. 2006) and the alligator EST assemblies from Chojnowski et al. (2007). The EnsEMBL sequences were annotations of the genomic sequences of the human, mouse, dog, gray short-tailed opossum, chicken, and western clawed frog and the set of these sequences that were identified by the turtle query were aligned using Clustal W (Chenna et al. 2003). Alignments that appeared largely anomalous, due to issues such as misannotations were discarded, as were individual sequences that appeared unlikely to be orthologs of the other sequences in the alignment or which exhibited anomalies. This increased the overall number of sequences analyzed but resulted in an unequal representation of species for each gene. Between 192 and 274 genes were included from each EnsEMBL species while 59 alligator genes were included from the dataset of Chojnowski et al. (2007). Alignments were optimized by eye using MacClade (Maddison and Maddison 2002) and the alignment segment present in all organisms (after anomalous sequences were discarded) was assigned to a CHARSET. Codon positions were then identified and PAUP* 4.0b10 (Swofford 2003) was used to calculate third-position base composition. Base composition data were extracted from the PAUP logfile using a shell script and imported into Microsoft Excel.
GC3 analyses
Strong correlations between GC3 of protein-coding genes, their introns, and their surrounding DNA (the isochores in which genes are embedded) have been found in both poikilotherms (Bernardi and Bernardi 1991) and homeotherms (Musto et al. 1999; Bernardi 2000). Thus, GC3 values for orthologous coding sequences were used as a surrogate to compare turtle isochore structure to that in other organisms, as in other studies (Zoubak et al. 1996; Bernardi et al. 1997; Galtier and Mouchiroud 1998). The open-source R statistical software (R Development Core Team 2007) was used to fit linear equations to the data using orthogonal regression (Isobe et al. 1990) and to calculate correlations. Lines for the best-fitting equation and for unity (y = x) are included in all plots.
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Types of genes analyzed
The genes obtained from the three embryonic turtle-subtraction libraries and their orthologs in other organisms exhibited a striking difference in GC3 from the set of genes obtained from a standard library (neither normalized nor subtracted) examined by Chojnowski et al. (2007
). Median GC3 values from all organisms were 
10% lower than in that study (Fig. 1) and the frog fell within the diversity of values for amniotes (albeit toward the lower end). This can be explained on the basis of the differences between the types of genes we expect to sample from the two different libraries, since there is strong evidence that the GC content of the genes is highly reflective of their type (e.g., housekeeping, specialized). The set of genes housed in GC-rich regions is greatly enriched for housekeeping genes and other highly expressed genes (Kudla et al. 2006; Arhondakis et al. 2008). Therefore, genes from GC-rich regions will be overrepresented in a standard library and genes from GC-poor regions are likely to be underrepresented. The opposite is expected for a subtracted library, where highly expressed genes are removed.
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To determine whether the difference in the types of genes represented in the libraries is a reasonable explanation for the lower overall median GC3 of the turtle ESTs, we calculated the median GC3 after restricting our analysis to the set of genes that are also present in the alligator (called "restricted" in Fig. 1). As we predicted, the restricted median GC3 values were almost identical to those reported in our previous study (Chojnowski et al. 2007
). Based upon the restricted median GC3 content, the turtle was the lowest of any amniote, indicating that the isochore structure of the turtle is intermediate between that of the frog and that seen in other amniotes.
The distribution of GC3 across organisms
The distribution of GC3 values, regardless of whether we consider the full set or the restricted set of sequences, also reveals substantial variation that is not captured by the median (Fig. 2). For example, the restricted median GC3 values for the frog and turtle differ by <1% but the proportion of genes in the highest GC3 category (90–100%) is different. In fact, the turtle GC3 distribution has a much greater skewness than does that of the frog (data not shown). These data are consistent with previous results (Bernardi 2000), indicating that amphibian genomes have a lower GC content than do amniote genomes. However, Bernardi (2000) also suggest that third-codon-position GC content distributions for genes in GC-poor regions, likely to have been enriched in our turtle libraries, show extensive overlap in different organisms.
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Turtle isochore structure is intermediate between those of amphibians and other amniotes
The relationship between the GC3 values for different organisms can be examined using orthogonal regression (Chojnowski et al. 2007
), and the slopes of lines obtained in this way reveal the degree of similarity between the isochore structures of the organisms analyzed. A slope of unity would indicate identical isochore structure (at least from the standpoint of GC3 content) and slopes with larger deviations from unity also correspond to organisms with greater differences in their isochore structures. Remarkably, the slopes of lines describing the relationships between organisms were very similar to those in our previous study (Chojnowski et al. 2007), despite using all set of turtle sequences rather than the restricted set. Thus, the genes sampled from the subtraction library do not differ from the genes sampled by Chojnowski et al. (2007) from the standpoint of the slope of these comparisons. In fact, comparisons between the turtle and various mammals revealed slopes that were both lower than unity and lower than in comparison between either turtle and archosaurs or between turtles and frogs (Fig. 3). Although the slopes of the comparisons between turtles and archosaurs were less than unity, the slope of those between frogs and turtles exceeds one. These results are consistent with a model in which the isochore structure of the turtle is intermediate between that of the frog and that of other amniotes.
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The correlation coefficient provides another way of examining the relationship between the isochore structures of two different organisms, since the number of genes that undergo changes in GC3 content may differ in distinct lineages. The turtle shows a higher correlation with the archosaurs than with any other organisms (Table 1). The lowest correlation coefficients involved comparisons with the frog, consistent with the divergent phylogenetic position of this organism (Fig. 4). However, there was also evidence for complex patterns of change in amniote isochore structures. For example, comparisons involving the opossum also show consistently low correlation coefficients (Table 1), with the exception of the comparison between opossums and mice. This is consistent with other studies showing a decrease in the GC content of the opossum (Gu and Li 2006
; Gu et al. 2007). Comparisons involving the opossum reveal a pattern that contrasts sharply with those involving the turtle. The latter, especially the comparisons between turtles and archosaurs, have high correlation coefficients but slopes that are less than unity. This suggests that turtle genes and archosaur genes tend to be similar in categories of GC3 content although the GC content of those categories differs, especially for the most GC-rich genes. Thus, few genes in the turtle–archosaur clades have undergone changes in their GC content category but the slopes of comparisons between turtles and archosaurs indicate that the most GC-rich genes have increased their GC content in archosaurs, decreased their GC content in turtles, or that both changes have occurred.
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A phylogenetic framework for reptilian isochore evolution
A single origin for GC-rich isochore structure at the base of the amniotes was the most parsimonious interpretation of the data on alligator isochore structure provided by Chojnowski et al. (2007
). This "amniote hypothesis" was suggested by a previous study that used a smaller set of turtle genes and crocodilian genes (Hughes et al. 1999), and it was embraced by studies that advocated nonthermal hypotheses for the origin of GC-rich isochores (Duret et al. 2002). The amniote hypothesis is inconsistent with the thermal models, since they predict two independent origins of GC-rich isochore structure coincident with the origin of homeothermy in birds and mammals (Bernardi 2000, 2007). A simple convergent origin of GC-rich isochore structure in mammals and birds is excluded by the existence of a GC-rich isochore structure in alligators (Chojnowski et al. 2007). The analyses presented here indicate that the isochore structure of turtles is intermediate between that of frogs and the GC-rich isochore structures of archosaurs and mammals. Thus, there are two hypotheses regarding the evolution of GC-rich isochores in amniotes, a convergent origin in mammals and archosaurs (combined with a modest increase in the GC content of the turtle genome), or an origin in the common ancestor of amniotes along with the loss of this GC-rich structure in turtles.
Chojnowski et al. (2007) suggested a modified thermal hypothesis, the "non-homeothermic thermal hypothesis," that is consistent with the GC-rich isochore structure of the alligator. This hypothesis postulates that GC-rich isochores reflect the maintenance of relatively high body temperatures either due to endothermy (in mammals and birds) or behavior and thermal inertial (in crocodilians) (Seebacher et al. 1999). Alternatively, extant crocodilians may have had homeothermic ancestors (Seymour et al. 2004). If this was the case, the GC-rich isochore structure of the alligator may be retained from an ancestral condition. However, there are a number of arguments against the hypothesis that ancestors of extant crocodilians were homeotherms (Hillenius and Ruben 2004), and we contend that behavioral thermoregulation is sufficient to explain the isochore structure of crocodilians if GC-rich isochore structure is related to thermal factors.
This study is consistent with a modified (nonhomeothermic) thermal hypothesis, since most turtles have less thermal inertia than do crocodilians. Indeed, the typical body temperature of the turtle species used in this study is lower than typical body temperatures of crocodilians (Gatten 1974; Seebacher et al. 1999), making it reasonable to postulate that turtles would have an isochore structure intermediate between those of amphibians and archosaurs were the thermal hypothesis correct. However, turtle thermoregulation is complex (Thomas et al. 1999) and it is likely to have undergone multiple changes over evolutionary times. Given the challenges associated with modeling behavioral thermoregulation for extant organisms (Christian et al. 2006) it will probably be very difficult to develop successful models able to be applied to the evolutionary history of extant organisms. Despite the challenges, it may be desirable to extend these analyses beyond the examination of reptiles to include the examination of isochore structures in homeothermic lineages that undergo torpor or hibernation, to determine whether the GC content underwent a decrease correlated with their fluctuating body temperatures.
Regardless of the specific relationship between isochore structure and thermal biology, or even whether any relationship of this type exists, expanding the amount of sequence data available for reptiles will be helpful. Varriale and Bernardi (2006) measured the GC contents of reptilian genomes using HPLC and analytical ultracentrifugation and they found only a modest difference between turtles and crocodilians. Shedlock et al. (2007) also reported similarities between the genomes of turtles and archosaurs. Varriale and Bernardi (2006) also noted substantial variation within lizards and snakes, suggesting that detailed analyses of the genomes of lizards and snakes will prove fruitful. A draft genome sequence for the lizard Anolis carolinensis will soon be available, so it will be possible to conduct detailed analyses of isochore structure for that organism. Our observation that there is a continuum of isochore structures within the reptiles indicates that exploring the variation in isochore structures within reptiles further will prove illuminating.
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These analyses of turtle EST data, especially when combined with previous analyses of sequence data from reptiles (Hughes et al. 1999
; Hamada et al. 2002; Chojnowski et al. 2007; Fortes et al. 2007), provides surprising information about the variance in the isochore structures of reptilian genomes. The turtle appears to have a relatively GC-rich isochore structure, stronger than the isochore structure of the frog but weaker than that of mammals or archosaurs. Our previous work emphasized the need to consider a complete set of models, which we defined based upon whether the models invoke patterns of mutation or selection and whether or not they involved thermal factors. If the complete set of models that are plausible a priori are considered, it should be possible to further constrain the credible set of models for isochore evolution or to provide the novel information necessary to define additional models that are better able to explain isochore evolution. |
Acknowledgments |
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We are grateful to Rebecca Kimball and the members of the Braun-Kimball lab group for helpful discussions, to Michael W. McCoy for help with R and statistical advice, and to Tamaki Yuri for suggesting additional analyses. This work was facilitated by grants from the National Science Foundation (DEB-0228682 to E.L.B., Rebecca T. Kimball, and David W. Steadman), the University of Florida Opportunity Fund (to E.L.B. and Louis J. Guillette Jr), and the American Society of Ichthyology and Herpetology (to J.L.C.).
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From the symposium "Reptile Genomics and Evolutionary Genetics" presented at the annual meeting of the Society for Integrative and Comparative Biology, January 2–6, 2008, at San Antonio, Texas.
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