Reptile Evolution and Genetics: An Overview.

The study of evolution has been indissolubly linked to the study of heredity since its inception [1]. [...].

Isolation and characterization of two satellite DNAs in Atlantolacerta andreanskyi (Werner, 1929) (Reptilia, Lacertidae).

Two satellite DNAs (satDNAs) have been isolated and characterized from three populations of Atlantolacerta andreanskyi. One satDNA (AAN-TaqI) has been isolated here from the first time. It is characterized by a tendency to AT enrichment (AT = 54.2%) and monomer length ranging from 187 to 199 bp. FISH experiments showed that this element occurs in subterminal position on the short arms of all chromosomes of the complement. The analyses of genetic variability of AAN-TaqI showed that the concerted evolution is acting effectively on these repeats that form separate clusters consistent with the geographic origin in the phylogenetic tree, thus supporting the hypothesis that A. andreanskyi would be a species complex. In addition, in the population from Jbel Aoulime this satDNA is already differentiated into two subfamilies. The other satDNA belongs to the family of IMO-TaqI already isolated in other lacertids. Differently from AAN-TaqI, concerted evolution does not seem to act effectively on this element that is not differentiated between populations. These results confirm that IMO-TaqI (AT = 53.4%) is conserved in both chromosomal position and most of its sequence in the lacertids from which it has been characterized so far. Its remarkable evolutionary conservation for about 45 million years could indicate that this satDNA may have a functional role that future investigations could unveil. Once again, this study shows how satDNAs coexisting in the same genome may differ in their evolutionary pattern, even though the reasons underlying this phenomenon in the species here studied have still to be fully understood.

Characterization of a satellite DNA in the genera Lacerta and Timon (Reptilia, Lacertidae) and its role in the differentiation of the W chromosome.

In this study, IMO-TaqI satDNA, previously isolated in several species of Lacertidae, was isolated and characterized from four species of the genus Lacerta and three of the genus Timon. The aim was to gain further insights into the evolutionary dynamics of this satDNA, its occurrence among lacertids and to understand if it plays any role in sex chromosome evolution in these seven species. The results here obtained highlighted the presence of this repetitive element in the genome of all the species investigated, thus indicating that IMO-TaqI satDNA is evolutionary conserved among a wide variety of lacertids. In addition, this element was found to be very abundant in the constitutive heterochromatin of the W-sex chromosome of the four Lacerta species investigated. The occurrence of IMO-TaqI satDNA on Lacerta heterochromosome suggests that it is involved in the differentiation of the W chromosome by heterochromatinization, and the fact that it is absent in the W of other lacertids investigated seems to confirm that repetitive DNA sequences would remain randomly trapped into the sex chromosomes, undergoing amplification as a consequence of the suppression of recombination.

Cytogenetic Characterization of a Population of Acanthodactylus lineomaculatus Duméril and Bibron, 1839 (Reptilia, Lacertidae), from Southwestern Morocco and Insights into Sex Chromosome Evolution.

Acanthodactylus lineomaculatus is now regarded as an ecotype of A. erythrurus with which it has been recently synonymized. Despite the wide range of A. erythrurus, karyological data for this species are scarce and limited to classical cytogenetic studies carried out in individuals from only 2 locations (central Spain and Spanish enclave of Melilla on the northwestern Mediterranean Moroccan coast). Here, for the first time, we cytogenetically characterized individuals of A. lineomaculatus from the southwestern Moroccan Atlantic coast with the aim to increase the karyological knowledge of this wide-ranging species and to assess if any chromosomal changes can be found in this ecotype in comparison to other populations of this species. The diploid number of the individuals investigated is 2n = 38 which is typical of most lacertids. Active NORs were located telomerically in a medium-small pair of chromosomes, and no inactive NORs were detected. C-banding revealed an intensely heterochromatic W chromosome composed of AT-rich (centromere and long arm telomeric region) and GC-rich (most of the long arm) regions, with extended interstitial telomeric sequences. These telomere-like repeats occupy the GC-rich heterochromatin of the W. The DNA composition of the W represents a trait distinguishing A. lineomaculatus (southwestern Morocco) from A. erythrurus from Spain that possess a DAPI-positive (AT-rich) W chromosome. In conclusion, these results add further evidence to the remarkable karyotype conservation in lacertid lizards, although differences in NOR location and in W chromosome structure among populations could suggest an incipient speciation mediated by chromosome changes in this wide-ranging lizard species.

The Lungfish Transcriptome: A Glimpse into Molecular Evolution Events at the Transition from Water to Land.

Lungfish and coelacanths are the only living sarcopterygian fish. The phylogenetic relationship of lungfish to the last common ancestor of tetrapods and their close morphological similarity to their fossil ancestors make this species uniquely interesting. However their genome size, the largest among vertebrates, is hampering the generation of a whole genome sequence. To provide a partial solution to the problem, a high-coverage lungfish reference transcriptome was generated and assembled. The present findings indicate that lungfish, not coelacanths, are the closest relatives to land-adapted vertebrates. Whereas protein-coding genes evolve at a very slow rate, possibly reflecting a "living fossil" status, transposable elements appear to be active and show high diversity, suggesting a role for them in the remarkable expansion of the lungfish genome. Analyses of single genes and gene families documented changes connected to the water to land transition and demonstrated the value of the lungfish reference transcriptome for comparative studies of vertebrate evolution.

Repetitive DNA in eukaryotic genomes.

Repetitive DNA--sequence motifs repeated hundreds or thousands of times in the genome--makes up the major proportion of all the nuclear DNA in most eukaryotic genomes. However, the significance of repetitive DNA in the genome is not completely understood, and it has been considered to have both structural and functional roles, or perhaps even no essential role. High-throughput DNA sequencing reveals huge numbers of repetitive sequences. Most bioinformatic studies focus on low-copy DNA including genes, and hence, the analyses collapse repeats in assemblies presenting only one or a few copies, often masking out and ignoring them in both DNA and RNA read data. Chromosomal studies are proving vital to examine the distribution and evolution of sequences because of the challenges of analysis of sequence data. Many questions are open about the origin, evolutionary mode and functions that repetitive sequences might have in the genome. Some, the satellite DNAs, are present in long arrays of similar motifs at a small number of sites, while others, particularly the transposable elements (DNA transposons and retrotranposons), are dispersed over regions of the genome; in both cases, sequence motifs may be located at relatively specific chromosome domains such as centromeres or subtelomeric regions. Here, we overview a range of works involving detailed characterization of the nature of all types of repetitive sequences, in particular their organization, abundance, chromosome localization, variation in sequence within and between chromosomes, and, importantly, the investigation of their transcription or expression activity. Comparison of the nature and locations of sequences between more, and less, related species is providing extensive information about their evolution and amplification. Some repetitive sequences are extremely well conserved between species, while others are among the most variable, defining differences between even closely relative species. These data suggest contrasting modes of evolution of repetitive DNA of different types, including selfish sequences that propagate themselves and may even be transferred horizontally between species rather than by descent, through to sequences that have a tendency to amplification because of their sequence motifs, to those that have structural significance because of their bulk rather than precise sequence. Functional consequences of repeats include generation of variability by movement and insertion in the genome (giving useful genetic markers), the definition of centromeres, expression under stress conditions and regulation of gene expression via RNA moieties. Molecular cytogenetics and bioinformatic studies in a comparative context are now enabling understanding of the nature and behaviour of this major genomic component.

Evolutionary dynamics of two satellite DNA families in rock lizards of the genus Iberolacerta (Squamata, Lacertidae): different histories but common traits.

Satellite DNAs compose a large portion of all higher eukaryotic genomes. The turnover of these highly repetitive sequences is an important element in genome organization and evolution. However, information about the structure and dynamics of reptilian satellite DNA is still scarce. Two satellite DNA families, HindIII and TaqI, have been previously characterized in four species of the genus Iberolacerta. These families showed different chromosomal locations, abundances, and evolutionary rates. Here, we extend the study of both satellite DNAs (satDNAs) to the remaining Iberolacerta species, with the aim to investigate the patterns of variability and factors influencing the evolution of these repetitive sequences. Our results revealed disparate patterns but also common traits in the evolutionary histories of these satellite families: (i) each satellite DNA is made up of a library of monomer variants or subfamilies shared by related species; (ii) species-specific profiles of satellite repeats are shaped by expansions and/or contractions of different variants from the library; (iii) different turnover rates, even among closely related species, result in great differences in overall sequence homogeneity and in concerted or non-concerted evolution patterns, which may not reflect the phylogenetic relationships among taxa. Contrasting turnover rates are possibly related to genomic constraints such as karyotype architecture and the interspersed organization of diverging repeat variants in satellite arrays. Moreover, rapid changes in copy number, especially in the centromeric HindIII satDNA, may have been associated with chromosomal rearrangements and even contributed to speciation within Iberolacerta.

Transcription of tandemly repetitive DNA: functional roles.

A considerable fraction of the eukaryotic genome is made up of satellite DNA constituted of tandemly repeated sequences. These elements are mainly located at centromeres, pericentromeres, and telomeres and are major components of constitutive heterochromatin. Although originally satellite DNA was thought silent and inert, an increasing number of studies are providing evidence on its transcriptional activity supporting, on the contrary, an unexpected dynamicity. This review summarizes the multiple structural roles of satellite noncoding RNAs at chromosome level. Indeed, satellite noncoding RNAs play a role in the establishment of a heterochromatic state at centromere and telomere. These highly condensed structures are indispensable to preserve chromosome integrity and genome stability, preventing recombination events, and ensuring the correct chromosome pairing and segregation. Moreover, these RNA molecules seem to be involved also in maintaining centromere identity and in elongation, capping, and replication of telomere. Finally, the abnormal variation of centromeric and pericentromeric DNA transcription across major eukaryotic lineages in stress condition and disease has evidenced the critical role that these transcripts may play and the potentially dire consequences for the organism.

A novel satellite DNA isolated in Pecten jacobaeus shows high sequence similarity among molluscs.

The aim of this work is to investigate the sequence conservation and the evolution of repeated DNA in related species. Satellite DNA is a component of eukaryotic genomes and is made up of tandemly repeated sequences. These sequences are affected by high rates of mutation that lead to the occurrence of species-specific satellite DNAs, which are different in terms of both quantity and quality. In this work, a novel repetitive DNA family, named PjHhaI sat, is described in Pecten jacobaeus. The quantitative analyses revealed a different abundance of this element in the molluscan species investigated in agreement with the "library hypothesis" even if, in this case, at a high taxonomic level. In addition, the qualitative analysis demonstrated an astonishing sequence conservation not only among scallops but also in six other molluscan species belonging to three classes. These findings suggest that the PjHhaI sat may be considered as the most ancients of DNA described so far, which remained "frozen" during molluscan evolution. The widespread distribution of this sat DNA in molluscs as well as its long evolutionary preservation open up questions on the functional role of this element. A future challenge might be the identification of proteins or molecules which interact with the PjHhaI sat.

Transposons, Genome Size, and Evolutionary Insights in Animals.

The relationship between genome size and the percentage of transposons in 161 animal species evidenced that variations in genome size are linked to the amplification or the contraction of transposable elements. The activity of transposable elements could represent a response to environmental stressors. Indeed, although with different trends in protostomes and deuterostomes, comprehensive changes in genome size were recorded in concomitance with particular periods of evolutionary history or adaptations to specific environments. During evolution, genome size and the presence of transposable elements have influenced structural and functional parameters of genomes and cells. Changes of these parameters have had an impact on morphological and functional characteristics of the organism on which natural selection directly acts. Therefore, the current situation represents a balance between insertion and amplification of transposons and the mechanisms responsible for their deletion or for decreasing their activity. Among the latter, methylation and the silencing action of small RNAs likely represent the most frequent mechanisms.

Characterization of purine catabolic pathway genes in coelacanths.

Coelacanths are a critically valuable species to explore the gene changes that took place in the transition from aquatic to terrestrial life. One interesting and biologically relevant feature of the genus Latimeria is ureotelism. However not all urea is excreted from the body; in fact high concentrations are retained in plasma and seem to be involved in osmoregulation. The purine catabolic pathway, which leads to urea production in Latimeria, has progressively lost some steps, reflecting an enzyme loss during diversification of terrestrial species. We report the results of analyses of the liver and testis transcriptomes of the Indonesian coelacanth Latimeria menadoensis and of the genome of Latimeria chalumnae, which has recently been fully sequenced in the framework of the coelacanth genome project. We describe five genes, uricase, 5-hydroxyisourate hydrolase, parahox neighbor B, allantoinase, and allantoicase, each coding for one of the five enzymes involved in urate degradation to urea, and report the identification of a putative second form of 5-hydroxyisourate hydrolase that is characteristic of the genus Latimeria. The present data also highlight the activity of the complete purine pathway in the coelacanth liver and suggest its involvement in the maintenance of high plasma urea concentrations.

Transcriptional activity of transposable elements in coelacanth.

The morphological stasis of coelacanths has long suggested a slow evolutionary rate. General genomic stasis might also imply a decrease of transposable elements activity. To evaluate the potential activity of transposable elements (TEs) in "living fossil" species, transcriptomic data of Latimeria chalumnae and its Indonesian congener Latimeria menadoensis were compared through the RNA-sequencing mapping procedures in three different organs (liver, testis, and muscle). The analysis of coelacanth transcriptomes highlights a significant percentage of transcribed TEs in both species. Major contributors are LINE retrotransposons, especially from the CR1 family. Furthermore, some particular elements such as a LF-SINE and a LINE2 sequences seem to be more expressed than other elements. The amount of TEs expressed in testis suggests possible transposition burst in incoming generations. Moreover, significant amount of TEs in liver and muscle transcriptomes were also observed. Analyses of elements displaying marked organ-specific expression gave us the opportunity to highlight exaptation cases, that is, the recruitment of TEs as new cellular genes, but also to identify a new Latimeria-specific family of Short Interspersed Nuclear Elements called CoeG-SINEs. Overall, transcriptome results do not seem to be in line with a slow-evolving genome with poor TE activity.

Isolation and characterization of two satellite DNAs in some Iberian rock lizards (Squamata, Lacertidae).

Satellite DNAs represent a large portion of all high eukaryotic genomes. They consist of numerous very similar repeated sequences, tandemly arranged in large clusters up to 100 million base pairs in length, usually located in the heterochromatic parts of chromosomes. The biological significance of satDNAs is still under discussion, but most of their proposed functions are related to heterochromatin and/or centromere formation and function. Because information about the structure of reptilian satDNA is far from exhaustive, we present a molecular and cytogenetic characterization of two satDNA families in four lacertid species. Two families of tandemly repeated DNAs, namely TaqI and HindIII satDNAs, have been cloned and sequenced from four species belonging to the genus Iberolacerta. These satDNAs are characterized by a monomer length of 171-188 and 170-172 bp, and by an AT content of 60.5% and 58.1%, respectively. FISH experiments with TaqI satDNA probe produced bright signals in pericentromeric regions of a subset of chromosomes whereas all the centromeres were marked by HindIII probe. The results obtained in this study suggest that chromosome location and abundance of satDNAs influence the evolution of these elements, with centromeric families evolving tenfold faster than interstitial/pericentromeric ones. Such different rates render different satellites useful for phylogenetic investigation at different taxonomic ranks.

Novel repeated DNAs in the antarctic polyplacophoran Nuttallochiton mirandus (Thiele, 1906).

Within the scope of a project on the characterization of satellite DNAs in polar mollusks, the Antarctic chiton Nuttallochitonmirandus (Thiele, 1906) was analyzed. Two novel families of tandemly repeated DNAs, namely NmH and NmP, are described in their structure and chromosomal localization, and, furthermore, their presence was analyzed in related species. Data reported here display a particular variability in the structural organization of DNA satellites within this species. Processes driving satellite evolution, which are likely responsible for the intriguing variability of the identified satellite DNAs, are discussed.

Analysis of the transcriptome of the Indonesian coelacanth Latimeria menadoensis.

BACKGROUND: Latimeria menadoensis is a coelacanth species first identified in 1997 in Indonesia, at 10,000 Km of distance from its African congener. To date, only six specimens have been caught and just a very limited molecular data is available. In the present work we describe the de novo transcriptome assembly obtained from liver and testis samples collected from the fifth specimen ever caught of this species. RESULTS: The deep RNA sequencing performed with Illumina technologies generated 145,435,156 paired-end reads, accounting for ~14 GB of sequence data, which were de novo assembled using a Trinity/CLC combined strategy. The assembly output was processed and filtered producing a set of 66,308 contigs, whose quality was thoroughly assessed. The comparison with the recently sequenced genome of the African congener Latimeria chalumnae and with the available genomic resources of other vertebrates revealed a good reconstruction of full length transcripts and a high coverage of the predicted full coelacanth transcriptome. CONCLUSION: Given the high genomic affinity between the two coelacanth species, the here described de novo transcriptome assembly can be considered a valuable support tool for the improvement of gene prediction within the genome of L. chalumnae and a valuable resource for investigation of many aspects of tetrapod evolution.

Characterization of sex determination and sex differentiation genes in Latimeria.

Genes involved in sex determination and differentiation have been identified in mice, humans, chickens, reptiles, amphibians and teleost fishes. However, little is known of their functional conservation, and it is unclear whether there is a common set of genes shared by all vertebrates. Coelacanths, basal Sarcopterygians and unique "living fossils", could help establish an inventory of the ancestral genes involved in these important developmental processes and provide insights into their components. In this study 33 genes from the genome of Latimeria chalumnae and from the liver and testis transcriptomes of Latimeria menadoensis, implicated in sex determination and differentiation, were identified and characterized and their expression levels measured. Interesting findings were obtained for GSDF, previously identified only in teleosts and now characterized for the first time in the sarcopterygian lineage; FGF9, which is not found in teleosts; and DMRT1, whose expression in adult gonads has recently been related to maintenance of sexual identity. The gene repertoire and testis-specific gene expression documented in coelacanths demonstrate a greater similarity to modern fishes and point to unexpected changes in the gene regulatory network governing sexual development.

Composition and phylogenetic analysis of vitellogenin coding sequences in the Indonesian coelacanth Latimeria menadoensis.

The coelacanth Latimeria menadoensis, a living fossil, occupies a key phylogenetic position to explore the changes that have affected the genomes of the aquatic vertebrates that colonized dry land. This is the first study to isolate and analyze L. menadoensis mRNA. Three different vitellogenin transcripts were identified and their inferred amino acid sequences compared to those of other known vertebrates. The phylogenetic data suggest that the evolutionary history of this gene family in coelacanths was characterized by a different duplication event than those which occurred in teleosts, amniotes, and amphibia. Comparison of the three sequences highlighted differences in functional sites. Moreover, despite the presence of conserved sites compared with the other oviparous vertebrates, some sites were seen to have changed, others to be similar only to those of teleosts, and others still to resemble only to those of tetrapods.

Conservation of chromosomes syntenic with avian autosomes in squamate reptiles revealed by comparative chromosome painting.

In contrast to mammals, birds exhibit a slow rate of chromosomal evolution. It is not clear whether high chromosome conservation is an evolutionary novelty of birds or was inherited from an earlier avian ancestor. The evolutionary conservatism of macrochromosomes between birds and turtles supports the latter possibility; however, the rate of chromosomal evolution is largely unknown in other sauropsids. In squamates, we previously reported strong conservatism of the chromosomes syntenic with the avian Z, which could reflect a peculiarity of this part of the genome. The chromosome 1 of iguanians and snakes is largely syntenic with chromosomes 3, 5 and 7 of the avian ancestral karyotype. In this project, we used comparative chromosome painting to determine how widely this synteny is conserved across nine families covering most of the main lineages of Squamata. The results suggest that the association of the avian ancestral chromosomes 3, 5 and 7 can be dated back to at least the early Jurassic and could be an ancestral characteristic for Unidentata (Serpentes, Iguania, Anguimorpha, Laterata and Scinciformata). In Squamata chromosome conservatism therefore also holds for the parts of the genome which are homologous to bird autosomes, and following on from this, a slow rate of chromosomal evolution could be a common characteristic of all sauropsids. The large evolutionary stasis in chromosome organization in birds therefore seems to be inherited from their ancestors, and it is particularly striking in comparison with mammals, probably the only major tetrapod lineage with an increased rate of chromosomal rearrangements as a whole.

Genome size, GC percentage and 5mC level in the Indonesian coelacanth Latimeria menadoensis.

The living fossil Latimeria menadoensis is important to understand sarcopterygian evolution. To gain further insights into this fish species we studied its genome size, GC% and 5mC level. The genome size and the GC% of the Indonesian coelacanth seem to be very similar to those of the African coelacanth. Moreover the GC%, the CpG frequency and the 5mC level of L. menadoensis are more similar to those of fish and amphibians than to those of mammals, birds and reptiles and this is in line with the hypothesis that two different DNA methylation and CpG shortage equilibria arose during vertebrate evolution. Our results suggest that the genome of L. menadoensis has remained unchanged for several million years, maybe since the origin of the lineage which from lobe-finned fish led to tetrapods. These data fit a conservative evolutionary landscape and suggest that the genome of the extant crossopterygians may be a sort of evolutionarily frozen genome.