Finding of Bov-B LINE retroelement in parthenogenetic and bisexual lizard species of the genus Darevskia (Lacertidae)

The Bov-B LINE retrotransposon was first discovered in Ruminantia and was long considered to be specific for this order. Later, this mobile element was described in snakes and some lizard species. Analysis of phylogenetic relationships of Bov-B LINE elements from different ruminants, snakes, and lizard species led to the suggestion on horizontal transfer of this retrotransposon from Squamata to Ruminantia. In the Squamata group, Bov-B LINE element was found in all snakes and some lizard species examined. The element was not detected in the genomes of some species of the genera Lacerta and Podarcis. In the present study, using PCR amplification and sequencing of PCR products, Bov-B LINE element was identified in the genomes of parthenogenetic and bisexual species of the genus Darevskia (Lacertidae), as well as in such species as Lacerta agilis and Zootoca vivipara, where this retrotransposon had not been not detected before.     

Short interspersed elements (SINEs) of squamate reptiles (Squam1 and Squam2): structure and phylogenetic significance

Short interspersed elements (SINEs) are important nuclear molecular markers of the evolution of many eukaryotes. However, the SINEs of squamate reptile genomes have been little studied. We first identified two families of SINEs, termed Squam1 and Squam2, in the DNA of meadow lizard Darevskia praticola (Lacertidae) by performing DNA hybridization and PCR. Later, the same families of retrotransposons were found using the same methods in members of another 25 lizard families (from Iguania, Scincomorpha, Gekkota, Varanoidea, and Diploglossa infraorders) and two snake families, but their abundances in these taxa varied greatly. Both SINEs were Squamata-specific and were absent from mammals, birds, crocodiles, turtles, amphibians, and fish. Squam1 possessed some characteristics common to tRNA-related SINEs from fish and mammals, while Squam2 belonged to the tRNA(Ala) group of SINEs and had a more unusual and divergent structure. Squam2-related sequences were found in several unannotated GenBank sequences of squamate reptiles. Squam1 abundance in the Polychrotidae, Agamidae, Leiolepididae, Chamaeleonidae, Scincidae, Lacertidae, Gekkonidae, Varanidae, Helodermatidae, and two snake families were 10(2) -10(4) times higher than those in other taxa (Corytophanidae, Iguanidae, Anguidae, Cordylidae, Gerrhosauridae, Pygopodidae, and Eublepharidae). A less dramatic degree of copy number variation was observed for Squam2 in different taxa. Several Squam1 copies from Lacertidae, Chamaeleonidae, Gekkonidae, Varanidae, and Colubridae were sequenced and found to have evident orthologous features, as well as taxa-specific autapomorphies. Squam1 from Lacertidae and Chamaeleonidae could be divided into several subgroups based on sequence differences. Possible applications of these SINEs as Squamata phylogeny markers are discussed.     

Finding of Bov-B LINE retroelement in parthenogenetic and bisexual lizard species of the genus Darevskia (Lacertidae)

The Bov-B LINE retrotransposon was first discovered in Ruminantia and was long considered to be specific for this order. Later, this mobile element was described in snakes and some lizard species. Analysis of phylogenetic relationships of Bov-B LINE elements from different ruminants, snakes, and lizard species led to the suggestion on horizontal transfer of this retrotransposon from Squamata to Ruminantia. In the Squamata group, Bov-B LINE element was found in all snakes and some lizard species examined. The element was not detected in the genomes of some species of the genera Lacerta and Podarcis. In the present study, using PCR amplification and sequencing of PCR products, Bov-B LINE element was identified in the genomes of parthenogenetic and bisexual species of the genus Darevskia (Lacertidae), as well as in such species as Lacerta agilis and Zootoca vivipara, where this retrotransposon had not been not detected before.     

Molecular phylogenetics of squamata: the position of snakes, amphisbaenians, and dibamids, and the root of the squamate tree

Squamate reptiles (snakes, lizards, and amphisbaenians) serve as model systems for evolutionary studies of a variety of morphological and behavioral traits, and phylogeny is crucial to many generalizations derived from such studies. Specifically, the traditional dichotomy between Iguania (anoles, iguanas, chameleons, etc.) and Scleroglossa (skinks, geckos, snakes, etc.) has been correlated with major evolutionary shifts within Squamata. We present a molecular phylogenetic study of 69 squamate species using approximately 4600 (2876 parsimony-informative) base pairs (bp) of DNA sequence data from the nuclear genes RAG-1(approximately 2750 bp) and c-mos(approximately 360 bp) and the mitochondrial ND2 region (approximately 1500 bp), sampling all major clades and most major subclades. Under our hypothesis, species previously placed in Iguania, Anguimorpha, and almost all recognized squamate families form strongly supported monophyletic groups. However, species previously placed in Scleroglossa, Varanoidea, and several other higher taxa do not form monophyletic groups. Iguania, the traditional sister group of Scleroglossa, is actually highly nested within Scleroglossa. This unconventional rooting does not seem to be due to long-branch attraction, base composition biases among taxa, or convergence caused by similar selective forces acting on nonsister taxa. Studies of functional tongue morphology and feeding mode have contrasted the similar states found in Sphenodon(the nearest outgroup to squamates) and Iguania with those of Scleroglossa, but our findings suggest that similar states in Sphenodonand Iguania result from homoplasy. Snakes, amphisbaenians, and dibamid lizards, limbless forms whose phylogenetic positions historically have been impossible to place with confidence, are not grouped together and appear to have evolved this condition independently. Amphisbaenians are the sister group of lacertids, and dibamid lizards diverged early in squamate evolutionary history. Snakes are grouped with iguanians, lacertiforms, and anguimorphs, but are not nested within anguimorphs.     

Ribosomal RNA genes in Scots pine (Pinus sylvestris L.): Chromosomal organization and structure

The nuclear 18S, 5.8S and 25S rRNA genes exist as thousands of rDNA repeats in the Scots pine genome. The number and location of rDNA loci (nucleolus organizers, NORs) were studied by cytological methods, and a restriction map from the coding region of the Scots pine rDNA repeat was constructed using digoxigenin-labeled flax rDNA as a probe. Based on the maximum number of nucleoli and chromosomal secondary constrictions, Scots pine has at least eight NORs in its haploid genome. The size of the Scots pine rDNA repeat unit is approximately 27 kb, two- or threefold larger than the typical angiosperm rDNA unit, but similar in size to other characterized conifer rDNA repeats. The intergenic spacer region (IGS) of the rDNA repeat unit in Scots pine is longer than 20 kb, and the transcribed spacer regions surrounding the 5.8S gene (ITS1 and ITS2) span a region of 2.9 kb. Restriction analysis revealed that although the coding regions of rDNA repeats are homogeneous, heterogeneity exists in the intergenic spacer region between individuals, as well as among the rDNA repeats within individuals.     

Functional and evolutionary morphology of lingual feeding in squamate reptiles: phylogenetics and kinematics

Use of the tongue as a prehensile organ during the ingestion stage of feeding in lizards was studied cinegraphically in seven species. Within Squamata, lingual prehension is limited to a single clade, the Iguania (Iguanidae, Agamidae and Chamaeleontidae), which includes all 'fleshy-tongued' lizards. All remaining squamates (Scleroglossa) use the jaws alone for prey prehension. Lingual prehension and a 'fleshy' tongue are primitive squamate characteristics. Kinematically, lingual ingestion cycles are similar to previously described transport cycles in having slow open, fast open, fast close and slow close-power stroke phases. Tongue movements are sequentially correlated with jaw movements as they are in transport. However, during ingestion, anterior movement of the tongue includes an extra-oral, as well as intra-oral component. Tongue protrusion results in a pronounced slow open-II phase at a large gape distance. A high degree of variability in quantitative aspects of ingestion and transport cycles suggests that modulation through sensory feedback is an important aspect of lizard feeding. Preliminary evidence indicates an important role for hyoid movement in tongue protrusion. Our results are consistent with the Bramble & Wake (1985) model generalized feeding cycle and support their contention that specialized feeding mechanisms often represent modifications of a basic pattern, particularly modification of the slow open phase.     

Morphological evolution of the lizard skull: a geometric morphometrics survey

Patterns of diversity among lizard skulls were studied from a morphological, phylogenetic, and functional perspective. A sample of 1,030 lizard skulls from 441 species in 17 families was used to create a lizard skull morphospace. This morphospace was combined with a phylogeny of lizard families to summarize general trends in the evolution of the lizard skull. A basal morphological split between the Iguania and Scleroglossa was observed. Iguanians are characterized by a short, high skull, with large areas of attachment for the external adductor musculature, relative to their sister group. The families of the Iguania appear to possess more intrafamilial morphological diversity than families of the Scleroglossa, but rarefaction of the data reveals this to be an artifact caused by the greater number of species represented in Iguanian families. Iguanian families also appear more dissimilar to one another than families of the Scleroglossa. Permutation tests indicate that this pattern is real and not due to the smaller number of families in the Iguanidae. Parallel and convergent evolution is observed among lizards with similar diets: ant and termite specialists, carnivores, and herbivores. However, these patterns are superimposed over the more general phylogenetic pattern of lizard skull diversity. This study has three central conclusions. Different clades of lizards show different patterns of disparity and divergence in patterns of morphospace occupation. Phylogeny imposes a primary signal upon which a secondary ecological signal is imprinted. Evolutionary patterns in skull metrics, taken with functional landmarks, allow testing of trends and the development of new hypotheses concerning both shape and biomechanics.     

Single-step co-integration of multiple expressible heterologous genes into the ribosomal DNA of the methylotrophic yeast Hansenula polymorpha

We have investigated the methylotrophic yeast Hansenula polymorpha as a host for the co-integration and expression of multiple heterologous genes using an rDNA integration approach. The ribosomal DNA (rDNA) of H. polymorpha was found to consist of a single rDNA cluster of about 50-60 repeats of an 8-kb unit located on chromosome II. A 2.4-kb segment of H. polymorpha rDNA encompassing parts of the 25S, the complete 5S and the non-transcribed spacer region between 25S and 18S rDNA was isolated and inserted into conventional integrative H. polymorpha plasmids harboring the Saccharomyces- cerevisiae-derived URA3 gene for selection. These rDNA plasmids integrated homologously into the rDNA repeats of a H. polymorpha (odc1) host as several independent clusters. Anticipating that this mode of multiple-cluster integration could be used for the simultaneous integration of several distinct rDNA plasmids, the host strain was co-transformed with a mixture of up to three different plasmids, all bearing the same URA3 selection marker. Transformations indeed resulted in mitotically stable strains harboring one, two, or all three plasmids integrated into the rDNA. The overall copy number of the plasmids integrated did not exceed the number of rDNA repeats present in the untransformed host strain, irrespective of the number of different plasmids involved. Strains harboring different plasmids co-expressed the introduced genes, resulting in functional proteins. Thus, this approach provides a new and attractive tool for the rapid generation of recombinant strains that simultaneously co-produce several proteins in desired stoichiometric ratios.     

Horizontal transfer of non-LTR retrotransposons in vertebrates

Since their discovery in family Bovidae (bovids), Bov-B LINEs, believed to be order-specific SINEs, have been found in all ruminants and recently also in Viperidae snakes. The distribution and the evolutionary relationships of Bov-B LINEs provide an indication of their origin and evolutionary dynamics in different species. The evolutionary origin of Bov-B LINE elements has been shown unequivocally to be in Squamata (squamates). The horizontal transfer of Bov-B LINE elements in vertebrates has been confirmed by their discontinuous phylogenetic distribution in Squamata (Serpentes and two lizard infra-orders) as well as in Ruminantia, by the high level of nucleotide identity, and by their phylogenetic relationships. The direction of horizontal transfer from Squamata to the ancestor of Ruminantia is evident from the genetic distances and discontinuous phylogenetic distribution of Bov-B LINE elements. The ancestral snake lineage (Boidae) has been recognized as a possible donor of Bov-B LINE elements to Ruminantia. The timing of horizontal transfer has been estimated from the distribution of Bov-B LINE elements in Ruminantia and the fossil data of Ruminantia to be 40–50mya. The phylogenetic relationships of Bov-B LINE elements from the various Squamata species agrees with that of the species phylogeny, suggesting that Bov-B LINE elements have been stably maintained by vertical transmission since the origin of Squamata in the Mesozoic era. This revised version was published online in July 2006 with corrections to the Cover Date.     

Monitoring the rate and dynamics of concerted evolution in the ribosomal DNA repeats of Saccharomyces cerevisiae using experimental evolution

Concerted evolution describes the unusual evolutionary pattern exhibited by certain repetitive sequences, whereby all the repeats are maintained in the genome with very similar sequences but differ between related species. The pattern of concerted evolution is thought to result from continual turnover of repeats by recombination, a process known as homogenization. Approaches to studying concerted evolution have largely been observational because of difficulties investigating repeat evolution in an experimental setting with large arrays of identical repeats. Here, we establish an experimental evolution approach to look at the rate and dynamics of concerted evolution in the ribosomal DNA (rDNA) repeats. A small targeted mutation was made in the spacer of a single rDNA unit in Saccharomyces cerevisiae so we could monitor the fate of this unit without the need for a selectable marker. The rate of loss of this single unit was determined, and the frequency of duplication was also estimated. The results show that duplication and deletion events occur at similar rates and are very common: An rDNA unit may be gained or lost as frequently as once every cell division. Investigation of the spatial dynamics of rDNA turnover showed that when the tagged repeat unit was duplicated, the copy predominantly, but not exclusively, ended up near to the tagged repeat. This suggests that variants in the rDNA spread in a semiclustered fashion. Surprisingly, large deletions that remove a significant fraction of total rDNA repeats were frequently found. We propose these large deletions are a driving force of concerted evolution, acting to increase homogenization efficiency over-and-above that afforded by turnover of individual rDNA units. Thus, the results presented here enhance our understanding of concerted evolution by offering insights into both the spatial and temporal dynamics of the homogenization process and suggest an important new aspect in our understanding of concerted evolution.     

Sclerotic Rings in Mosasaurs (Squamata: Mosasauridae): Structures and Taxonomic Diversity

Mosasaurs (Squamata: Mosasauridae) were a highly diverse, globally distributed group of aquatic lizards in the Late Cretaceous (98–66 million years ago) that exhibited a high degree of adaptation to life in water. To date, despite their rich fossil record, the anatomy of complete mosasaur sclerotic rings, embedded in the sclera of the eyeball, has not been thoroughly investigated. We here describe and compare sclerotic rings of four mosasaur genera, Tylosaurus, Platecarpus, Clidastes, and Mosasaurus, for the first time. Two specimens of Tylosaurus and Platecarpus share an exact scleral ossicle arrangement, excepting the missing portion in the specimen of Platecarpus. Furthermore, the exact arrangement and the total count of 14 ossicles per ring are shared between Tylosaurus and numerous living terrestrial lizard taxa, pertaining to both Iguania and Scleroglossa. In contrast, two species of Mosasaurus share the identical count of 12 ossicles and the arrangement with each other, while no living lizard taxa share exactly the same arrangement. Such a mosaic distribution of these traits both among squamates globally and among obligatorily aquatic mosasaurs specifically suggests that neither the ossicle count nor their arrangement played major roles in the aquatic adaptation in mosasaur eyes. All the mosasaur sclerotic rings examined consistently exhibit aperture eccentricity and the scleral ossicles with gently convex outer side. Hitherto unknown to any squamate taxa, one specimen of Platecarpus unexpectedly shows a raised, concentric band of roughened surface on the inner surface of the sclerotic ring. It is possible that one or both of these latter features may have related to adaptation towards aquatic vision in mosasaurs, but further quantitative study of extant reptilian clades containing both terrestrial and aquatic taxa is critical and necessary in order to understand possible adaptive significances of such osteological features.     

Structure of the central spacer region of extrachromosomal ribosomal DNA in Physarum polycephalum

We have analyzed the sequence organization of the central spacer region of the extrachromosomal ribosomal DNA from two strains of the acellular slime mold Physarum polycephalum. It had been inferred previously from electron microscopy that this region, which comprises about one third of the 60 kb³ palindromic rDNA, contains a complex series of inverted repetitious sequences. By partial digestion of end-labeled fragments isolated from purified rDNA and from rDNA fragments cloned in Escherichia coli, we have constructed a detailed restriction map of this region. The 11 kb of spacer DNA of each half molecule of rDNA contains the following elements: (a) two separate regions, one of 1.1 kb and one of 2.1 kb, composed of many direct repeats of the same 30 base-pair unit; (b) a region of 4.4 kb composed of a complex series of inverted repeats of a 310 base-pair unit; (c) another region of 1.6 kb composed of inverted repeats of the same 310 base-pair unit located directly adjacent to the center of the rDNA; (d) two copies of a unique sequence of 0.85 kb, which probably contains a replication origin. Some of the CpG sequences in the spacer resist cleavage by certain restriction endonucleases and thus appear to be methylated. The lack of perfect symmetry about the central axis and the arrangement of inverted repeated sequences explain the complex pattern of branches and forks of the fold-back molecules previously observed by electron microscopy. Comparison of the rDNA restriction maps from the two strains of Physarum suggests that the repeat units in the spacer are undergoing concerted evolution. We propose a model to explain the evolutionary origin of the several palindromic axes in the Physarum rDNA spacer.     

Lipophilic compounds in femoral secretions of male collared lizards,Crotaphytus bicinctores (Iguania,Crotaphytidae)

In many lizards, chemical compounds from the femoral gland secretions are used in intraspecific communication, but most studies describing these chemicals are for lizard species included in the Scleroglossa clade, whereas lizards within the Iguanian clade have been much less studied, probably because these lizards were considered to rely more on visual cues. However, many iguanian lizards have abundant femoral secretions and are able of chemosensory conspecific recognition, which might be based on compounds secreted by femoral glands. By using GC–MS analyses, we found 58 lipophilic compounds in femoral gland secretions of male Great Basin collared lizard, Crotaphytus bicinctores (Iguania, Crotaphytidae). Main compounds were steroids (mainly two triunsaturated steroids and cholesterol), carboxylic acids (mainly hexadecanoic acid), waxy esters of long chain fatty acids, alcohols (mainly hexadecanol), aldehydes and other minor compounds. We compared these compounds with those found in other lizard species and discussed the potential signaling function of some compounds and how the xeric habitat of this lizard could have conditioned the composition of secretions.     

Evolution of target specificity in R1 clade non-LTR retrotransposons

Although most non-long terminal repeat (non-LTR) retrotransposons are inserted throughout the host genome, many non-LTR elements in the R1 clade are inserted into specific sites within the target sequence. Four R1 clade families have distinct target specificity: R1 and RT insert into specific sites of 28S rDNA, and TRAS and SART insert into different sites within the (TTAGG)(n) telomeric repeats. To study the evolutionary history of target specificity of R1-clade retrotransposons, we have screened extensively novel representatives of the clade from various insects by in silico and degenerate polymerase chain reaction (PCR) cloning. We found four novel sequence-specific elements; Waldo (WaldoAg1, 2, and WaldoFs1) inserts into ACAY repeats, Mino (MinoAg1) into AC repeats, R6 into another specific site of the 28S rDNA, and R7 into a specific site of the 18S rDNA. In contrast, several elements (HOPE, WISHBm1, HidaAg1, NotoAg1, KagaAg1, Ha1Fs1) lost target sequence specificity, although some of them have preferred target sequences. Phylogenetic trees based on the RT and EN domains of each element showed that (1) three rDNA-specific elements, RT, R6, and R7, diverged from Waldo; (2) the elements having similar target sequences are phylogenetically related; and (3) the target specificity in the R1 clade was obtained once and thereafter altered and lost several times independently. These data indicate that the target specificity in R1 clade retroelements has changed during evolution and is more divergent than has been speculated so far.     

Molecular evolution of Bov-B LINEs in vertebrates

Since their discovery in family Bovidae (bovids), Bov-B LINEs, believed to be order-specific SINEs, have been found in all ruminants and recently also in Viperidae snakes. The distribution and the evolutionary relationships of Bov-B LINEs provide an indication of their origin and evolutionary dynamics in different species. The evolutionary origin of Bov-B LINE elements has been shown unequivocally to be in Squamata (squamates). The horizontal transfer of Bov-B LINE elements in vertebrates has been confirmed by their discontinuous phylogenetic distribution in Squamata (Serpentes and two lizard infra-orders) as well as in Ruminantia, by the high level of nucleotide identity, and by their phylogenetic relationships. The direction of horizontal transfer from Squamata to the ancestor of Ruminantia is evident from the genetic distances and discontinuous phylogenetic distribution of Bov-B LINE elements. The ancestor of Colubroidea snakes has been recognized as a possible donor of Bov-B LINE elements to Ruminantia. The timing of horizontal transfer has been estimated from the distribution of Bov-B LINE elements in Ruminantia and the fossil data of Ruminantia to be 40-50 My ago. The phylogenetic relationships of Bov-B LINE elements from the various Squamata species agrees with that of the species phylogeny, suggesting that Bov-B LINE elements have been stably maintained by vertical transmission since the origin of Squamata in the Mesozoic era.     

Two 5S rDNA arrays in Neotropical fish species: is it a general rule for fishes?

In this paper we describe Southern blot hybridization results probed with 5S rRNA genes for several Neotropical fish species representing different taxonomic groups. All the studied species showed a general trend with the 5S rDNA tandem repeats organized in two distinct size-classes. At the same time, data on 5S rDNA organization in fish genome were summarized. Previous information on the organization and evolution of 5S rRNA gene arrays in the genome of this vertebrate group are in agreement with the Southern results here presented. Sequences obtained for several fish species have revealed the occurrence of two distinct 5S rDNA classes characterized by distinct non-transcribed spacer sequences, which are clustered in different chromosomes in some species. Moreover, the 5S rDNA loci are generally distributed in an interstitial position in the chromosomes and they are usually not syntenic to the 45S rDNA. The presence of two classes of 5S rDNA in several non-related fish species suggests that this could be a common condition for the 5S rRNA gene organization in the fish genome.     

Genetic mapping of 18s ribosomal RNA-related loci to mouse Chromosomes 5, 6, 9, 12, 17, 18, 19, and X

The organization of ribosomal RNA genes (rDNA) in the genome of the mouse varies significantly from one strain to another, but has been shown to follow the pattern of clusters of tandem repeats located at chromosome ends, often associated with cytological nucleolus organizer regions. The number of copies of the repeat unit at each locus also varies. A probe for the 18S ribosomal RNA sequence on Southern blots reveals both high copy number bands and fainter bands indicative of low repeat number. We have mapped a number of newly identified low-copy-number rDNA loci in C57BL/6J, in addition to placing some of the NOR-associated rDNA repeats on the Jackson interspecific backcross (BSS) map. We suggest that additional low-copy-number loci may remain to be mapped, and that the evolution of rDNA loci in the genome may include the proliferation of single copies by retroinsertion or other mechanisms. Received: 23 February 1996 / Accepted: 29 July 1996     

Telomere and 45S rDNA sequences are structurally linked on the chromosomes in <Emphasis Type="Italic">Chrysanthemum segetum</Emphasis> L.

Some reports have shown that nucleolar organizer regions are located at the telomeric region and have a structural connection with telomeres at the cellular level in many organisms. In this study, we found that all 45S ribosomal DNA (rDNA) signals were located at telomeric regions on the chromosomes in Chrysanthemum segetum L., and the 45S rDNA showed distinct signal patterns on different metaphase chromosome spreads. The bicolor fluorescence in situ hybridization experiment on the extended fibers revealed that telomere repeats were structurally connected with or interspersed into rDNA sequences. The close cytological structure relation between rDNA and telomere sequences led us to use PCR with combinations of the telomere primer and the rDNA primer to obtain some fragments, which were flanked by different rDNA and telomere primer sequences. One representative clone CHS2 contains closely connected rDNA and telomere sequences, suggesting that the telomere sequence invaded into the conserved rDNA sequence. In addition, the sequences of some PCR clones were flanked by the single telomeric primer sequence or the rDNA primer sequence. These results suggested that homologous recombination occurred between tandem repeat units of rDNA sequences or telomere repeats at the chromosome terminus.     

Repeated DNA sequences found in the large spacer of Vicia faba rDNA

In the large spacer of the rDNA of Vicia faba, multiples of a 0.32 kilobasepair (kb) sequence reiterate to various degrees. We sequenced the repetitious region consisting of the repeating sequences and its flanking regions using two cloned plasmids, which contain V. faba rDNA segments encompassing the whole region of the large spacer. The repetitious region was found to consist of multiple complete copies and one truncated copy of a 325 bp repeat unit and to be flanked by direct repeat sequences of about 150 bp. The set of direct repeats located at either side of the repetitious region differed from each other with about 10% sequence heterogeneity. However, nucleotide sequences of the direct repeats were well conserved between the two clones examined. Southern blot hybridization indicated a widespread distribution within the whole V. faba genome of some related sequences with high homologies to the 325 bp repeat unit and to the direct repeats.