Variability of Restriction Sites in Satellite DNA as a Molecular Basis of Taxonprint Method: Evidence from the Study of Caucasian Rock Lizards

The distribution of restriction sites in satellite DNA of 17 Caucasian rock lizard species of the genus Lacerta (Darevskia gen. nov., (Squamata, Lacertidae) was analyzed. The distribution patterns were shown to reflect the degree of satellite DNA evolutionary divergence, which could be revealed by taxonprint method, i.e., through the analysis of genomic DNA with a set of restriction endonucleases and subsequent computer-aided analysis. Thus, the taxonprint method offers an opportunity to examine the satellite DNA divergence in closely related species and infer the phylogeny of the species studied without reserting to costly and labor-consuming procedures. This is the advantage of using this technique at the early stages phylogenetic analysis of genomic DNA for rapid and effective estimation of relationships between closely related species as well as in the cases when DNA cloning and sequencing are too expensive or not feasible.     

Restriction Endonuclease Analysis of Highly Repetitive DNA as a Phylogenetic Tool

Multiple band patterns of DNA repeats in the 20–500-nucleotide range can be detected by digesting genomic DNA with short—cutting restriction endonucleases, followed by end labeling of the restriction fragments and fractionation in nondenaturing polyacrylamide gels. We call such band patterns obtained from genomic DNA ``taxonprints' (Fedorov et al. 1992). Here we show that taxonprints for the taxonomic groups studied (mammals, reptiles, fish, insects—altogether more than 50 species) have the following properties: (1) All individuals from the same species have identical taxonprints. (2) Taxonprint bands can be subdivided into those specific for a single species and those specific for groups of closely related species, genera, and even families. (3) Each restriction endonuclease produces unique band patterns; thus, five to ten restriction enzymes (about 100 bands) may be sufficient for a statistical treatment of phylogenetic relationships based on polymorphisms of restriction endinuclease sites. We demonstrate that taxonprint analysis allows one to distinguish closely related species and to establish the degree of similarity among species and among genera. These characteristics make taxonprint analysis a valuable tool for taxonomic and phylogenetic studies. Received: 10 February 1997 / Accepted: 10 March 1997     

Variable and Invariable DNA Repeat Characters Revealed by Taxonprint Approach Are Useful for Molecular Systematics

A specially optimized restriction analysis of highly repetitive DNA elements, called DNA taxonprint, was applied for phylogenetic study of primates and lizards. It was shown that electrophoretic bands of DNA repeats revealed by the taxonprint technique have valuable properties for molecular systematics. Approximately half of taxonprint bands (TB) are invariable and do not disappear from the genomes during evolution or change spontaneously. Presumably these invariable bands are restriction fragments of dispersed DNA repeats. Another group represents variable taxonprint bands that differ even between closely related species. These variable bands are probably represented by tandem DNA repeats and could be used as species-specific markers. It was shown that taxonprint bands are independent characters since the appearance of a new taxonprint band does not change the previous band pattern. Phylogenetic reconstruction carried out on taxonprint data demonstrated that this approach could be of general utility for molecular systematics and species identification. Received: 12 January 1998 / Accepted: 16 May 1998     

Interspezifische Variabilität bei hypotrichen Ciliaten1

Interspecific variability in hypotrichous ciliates The genome organization of hypotrichous ciliates differs fundamentally from those of most other eukaryotic organisms. Every cell has two kinds of nuclei as is characteristic for ciliatese small generative micronuclei (Mi) whose DNA has a high molecular weight and which is organized in chromosomes, and vegetative macronuclei (Ma) which are very rich in DNA. The macronuclear DNA consists of so-called “gene-sized” DNA pieces, an organization which is not found in any other organism. This extraordinary genome organization offers a convenient experimental approach for studying evolutionary divergence at different molecular levels: 1. whole genomes, 2. subfractions of genomes, and 3. enzyme proteins. The comparison of unfractionated genomic DNA of hypotrichous ciliates by Dna-DNA hybridizations has yielded an unsuspected result: species that are closely related according to their morphology show an unusually low amount of sequence homology. The underlying reason might be that hypotrichous species separated early in eukaryotic evolution. Whereas the morphology of “closely related” species has changed only little, molecular evolution has led to major genomic changes that reflect the great evolutionary age of the species. The separation of native macronuclear DNA by gel electrophoresis produces species-specific DNA banding patterns based on different copy numbers of individual “gene-sized” DNA pieces in different species. These banding patterns allow the discrimination of sibling species which are morphologically very similar or even undistinguishable. Higher taxa can also be identified by means of DNA banding patterns. Cloned α- and β-tubulin genes were used in hybridization experiments to study the evolutionary divergence of individual DNA sequences in different hypotrichous species. The unusual Magenome organization makes such an analysis especially convenient. Characteristics of individual genes such as length number of sequence variants, copy number, and pattern of restriction sites can be compared with this method. The digestion of Mi-DNA with restriction endonucleases reveals differences in the repetitive DNA fraction of those genomes. Specific differences can be detected between closely related species and even between different populations of one species. The comparison of evolutionary divergence at the DNA level was supplemented by a comparison at the protein level. Enzyme electrophoresis proved to be a suitable method for the identification of otherwise indistinguishable species. Genetic ivergency (D-values) was estimated on the basis of allozyme data and a dendrogram was constructed reflecting the amount of genetic similarity between the species investigated. The discussion considers advantages and disadvantages of molecular characteristics for attacking taxonomic, phylogenetic, and evolutionary problems.     

The pattern of restriction enzyme-induced banding in the chromosomes of chimpanzee,gorilla, and orangutan and its evolutionary significance

Summary The pattern of banding induced by five restriction enzymes in the chromosome complement of chimpanzee, gorilla, and orangutan is described and compared with that of humans. The G banding pattern induced by Hae III was the only feature common to the four species. Although hominid species show almost complete chromosomal homology, the restriction enzyme C banding pattern differed among the species studied. Hinf I did not induce banding in chimpanzee chromosomes, and Rsa I did not elicit banding in chimpanzee and orangutan chromosomes. Equivalent amounts of similar satellite DNA fractions located in homologous chromosomes from different species or in nonhomologous chromosomes from the same species showed different banding patterns with identical restriction enzymes. The great variability in frequency of restriction sites observed between homologous chromosome regions may have resulted from the divergence of primordial sequences changing the frequency of restriction sites for each species and for each chromosomal pair. A total of 30 patterns of banding were found informative for analysis of the hominid geneaalogical tree. Using the principle of maximum parsimony, our data support a branching order in which the chimpanzee is more closely related to the gorilla than to the human.     

Inferring the mode of speciation from genomic data: a study of the great apes

The strictly allopatric model of speciation makes definable predictions on the pattern of divergence, one of which is the uniformity in the divergence time across genomic regions. Using 345 coding and 143 intergenic sequences from the African great apes, we were able to reject the null hypothesis that the divergence time in the coding sequences (CDSs) and intergenic sequences (IGSs) is the same between human and chimpanzee. The conclusion is further supported by the analysis of whole-genome sequences between these species. The difference suggests a prolonged period of genetic exchange during the formation of these two species. Because the analysis should be generally applicable, collecting DNA sequence data from many genomic regions between closely related species should help to settle the debate over the prevalence of the allopatric mode of speciation.     

Comparative genomics and repetitive sequence divergence in the species of diploid Nicotiana section Alatae

Combining phylogenetic reconstructions of species relationships with comparative genomic approaches is a powerful way to decipher evolutionary events associated with genome divergence. Here, we reconstruct the history of karyotype and tandem repeat evolution in species of diploid Nicotiana section Alatae. By analysis of plastid DNA, we resolved two clades with high bootstrap support, one containing N. alata, N. langsdorffii, N. forgetiana and N. bonariensis (called the n = 9 group) and another containing N. plumbaginifolia and N. longiflora (called the n = 10 group). Despite little plastid DNA sequence divergence, we observed, via fluorescent in situ hybridization, substantial chromosomal repatterning, including altered chromosome numbers, structure and distribution of repeats. Effort was focussed on 35S and 5S nuclear ribosomal DNA (rDNA) and the HRS60 satellite family of tandem repeats comprising the elements HRS60, NP3R and NP4R. We compared divergence of these repeats in diploids and polyploids of Nicotiana. There are dramatic shifts in the distribution of the satellite repeats and complete replacement of intergenic spacers (IGSs) of 35S rDNA associated with divergence of the species in section Alatae. We suggest that sequence homogenization has replaced HRS60 family repeats at sub-telomeric regions, but that this process may not occur, or occurs more slowly, when the repeats are found at intercalary locations. Sequence homogenization acts more rapidly (at least two orders of magnitude) on 35S rDNA than 5S rDNA and sub-telomeric satellite sequences. This rapid rate of divergence is analogous to that found in polyploid species, and is therefore, in plants, not only associated with polyploidy.     

Evolutionary dynamics of a satellite DNA in the tiger beetle species pair Cicindela campestris and C. maroccana.

Satellite repeat elements are an abundant component of eukaryotic genomes, but not enough is known about their evolutionary dynamics and their involvement in karyotype and species differentiation. We report the nucleotide sequence, chromosomal localization, and evolutionary dynamics of a repetitive DNA element of the tiger beetle species pair Cicindela maroccana and Cicindela campestris. The element was detected after restriction digest of C. maroccana total genomic DNA with EcoRI as a single band and its multimers on agarose gels. Cloning and sequencing of several isolates revealed a consensus sequence of 383 bp with no internal repeat structure and no detectable similarity to any entry in GenBank. Hybridization of the satellite unit to C. maroccana mitotic and meiotic chromosomes revealed the presence of this repetitive DNA in the centromeres of all chromosomes except the Y chromosome, which exhibited only a very weak signal in its short arm. PCR-based tests for this satellite in related species revealed its presence in the sister species C. campestris, but not in other closely related species. Phylogenetic analysis of PCR products revealed well-supported clades that generally separate copies from each species. Because both species exhibit the multiple X chromosome karyotypic system common to Cicindela, but differ in their X chromosome numbers (four in C. maroccana vs. three in C. campestris), structural differences could also be investigated with regard to the position of satellites in a newly arisen X chromosome. We find the satellite in a centromeric position in all X chromosomes of C. maroccana, suggesting that the origin of the additional X chromosome involves multiple karyotypic rearrangements.     

Concerted evolution of light satellite DNA in genus Mus implies amplification and homogenization of large blocks of repeats

Light satellite DNA components present in species belonging to the genus Mus and to related murids were studied using the Southern blot technique. The results show species variations in both the amount and periodic structure of the repeating units, which suggests that families of related higher-order repeats developed in a common ancestor and were then amplified and/or deleted to different extents during the subsequent evolutionary period. Although the patterns generated by a series of type B enzymes (restriction enzymes that possess sites in a limited number of segments making up the total satellite DNA) in the species closely related to the M. musculus complex were very similar, sequence analysis of cloned unit repeats in two of these species (M. musculus domesticus and M. spretoides) showed near fixation of species-diagnostic variant nucleotides. This suggests that the important amplification and homogenization events that occurred after the divergence of M. spretus must have involved large blocks of sequences.     

Chromosomal distribution and organization of three cervid satellite DNAs in Chinese water deer (Hydropotes inermis)

The species-specific profile and centromeric heterochromatin localization of satellite DNA in mammalian genomes imply that satellite DNA may play an important role in mammalian karyotype evolution and speciation. A satellite III DNA family, CCsatIII was thought to be specific to roe deer (Capreolus capreolus). In this study, however, this satellite DNA family was found also to exist in Chinese water deer (Hydropotes inermis) by PCR-Southern screening. A satellite III DNA element of this species was then generated from PCR-cloning by amplifying this satellite element using primer sequences from the roe deer satellite III clone (CCsatIII). The newly generated satellite III DNA along with previously obtained satellite I and II DNA clones were used as probes for FISH studies to investigate the genomic distribution and organization of these three satellite DNA families in centromeric heterochromatin regions of Chinese water deer chromosomes. Satellite I and II DNA were observed in the pericentric/centric regions of all chromosomes, whereas satellite III was distributed on 38 out of 70 chromosomes. The distribution and orientation of satellite DNAs I, II and III in the centromeric heterochromatin regions of the genome were further classified into four different types. The existence of a Capreolus-like satellite III in Chinese water deer implies that satellite III is not specific to the genus Capreolus (Buntjer et al., 1998) and supports the molecular phylogeny classification of Randi et al. (1998) which suggests that Chinese water deer and roe deer are closely related.     

Mitochondrial DNA evolution in the Montium-species subgroup of Drosophila

Mitochondrial DNA (mtDNA) restriction-site maps for six species (10 strains) of the Drosophila montium subgroup were established. A total of 50 restriction sites were mapped, corresponding to 1.67% of the mtDNA genome. On the basis of differences in the restriction sites, nucleotide divergence (delta) was calculated for each pair of species (strains), and phylogenetic trees were constructed by using distance- matrix and parsimony methods. Comparison of the resultant phylogenetic trees shows that the sibling species D. auraria and D. quadraria are closely related. At the other extreme, considerable divergence was observed between the two strains of D. serrata and between D. serrata and D. birchii, a finding that contrasts with their grouping within the same species complex. Nevertheless, our data indicate that these six oriental montium species are rather closely related.      

PHYLOGENETIC IMPLICATIONS OF CHLOROPLAST DNA RESTRICTION SITE VARIATION IN THE MUTISIEAE (ASTERACEAE)

Phylogenetic relationships among 13 species in the tribe Mutisieae and a single species from each of three other tribes in the Asteraceae were assessed by chloroplast DNA restriction site mapping. Initially, 211 restriction site mutations were detected among 16 species using 10 restriction enzymes. Examination of 12 of these species using nine more enzymes revealed 179 additional restriction site mutations. Phylogenetic analyses of restriction site mutations were performed using both Dolio and Wagner parsimony, and the resulting monophyletic groups were statistically tested by the bootstrap method. The phylogenetic trees confirm an ancient evolutionary split in the Asteraceae that was previously suggested by the distribution of a chloroplast DNA inversion. The subtribe Barnadesiinae of the tribe Mutisieae is shown to be the ancestral group within the Asteraceae. The molecular phylogenies also confirm the paraphyly of the Mutisieae and provide statistical support for the monophyly of three of its four currently recognized subtribes (Barnadesiinae, Mutisiinae, and Nassauviinae). The fourth subtribe, Gochnatiinae, is shown to be paraphyletic. Within the subtribes, several closely related generic pairs are identified. Chloroplast DNA sequence divergence among genera of the Asteraceae ranges between 0.7 and 5.4%, which is relatively low in comparison to other angiosperm groups. This suggests that the Asteraceae is either a relatively young family or that its chloroplast DNA has evolved at a slower rate than in other families.     

Organization of a dispersed repeated DNA element in the Zamia genome

Occurrence and genomic organization of dispersed elements containing ZpS1 satellite repeats have been investigated in a wide representation of species of the old plant genus Zamia (Zamiaceae, Cycadales). In Z. paucijuga, the ZpS1 repeat is organized as long satellite DNA arrays and as short arrays inserted into AT-rich dispersed elements. A comparative study by Southern analysis shows that these unusual dispersed elements containing the ZpS1 repeat are present with different organizations in all investigated Zamia species. In some species these elements are present with a low copy number, while in other species secondary amplification events, involving specific sequence clusters, appear to have generated characteristic dispersed elements in a high copy number. Among Zamia species, several groups share similar restriction patterns, as the Zamia loddigesii complex and the Caribbean species suggesting a general correlation between organization and genomic representation of the dispersed repeated sequence and the pattern of phyletic relationships in the genus. However, the finding of different patterns also among closely related species suggests a complex history of amplifications and losses of these dispersed repetitive elements that cannot be always easily traced through the phylogenetic reconstruction of this ancient plant group.     

Phylogenetic relationships among the subfamily Coregoninae as revealed by mitochondrial DNA restriction analysis

Mitochondria1 DNA (mtDNA) restriction analysis was used to assess phylogenetic patterns among 21 taxa of the subfamily Coregoninae. The genus Prosopium formed a very distinct group differing by 10% (sequence divergence estimate) from other species. Coregonus and Stenodus species were closely related, diverging by sequence divergence estimates of less than 5.6%. These species split into two major sister groups. One comprised all 'true whitefish' (subgenus Coregonus ) and four cisco species (subgenus Leucichrhys ). The most distant species within this assemblage was the Acadian whitefish ( C. huntsmani ). The other group included all other cisco species and also the Inconnu ( Stenodus leucichthys ). These results supported a polyphyletic origin of the ciscoes, and did not support Stenodus as a sister taxon of the genus Coregonus . The levels of sequence divergence observed suggested that most extant coregonines radiated during the Pleistocene.     

Taxonomic and molecular studies on Drosophila sinobscura and D. hubeiensis,two sibling species of the D. obscura group*

Allozyme electrophoresis and three different DNA sequences (ATOC180 satellite DNA, 5SrDNA repeats, and parts of the Adh gene) were used to compare the two closely related East Asian sibling species Drosophila sinobscura and D. hubeiensis producing fertile hybrids in the laboratory. The data were also applied to establish their phylogenetic relationships to the other species of the D. obscura group. Genetic divergence in 5SrDNA repeats and specifically in the Adh gene separate the two species clearly from each other and justify their species status. Both species are related to the European species of the D. obscura group but the presence of members of the ATOC 180 satellite DNA family, specific and common to the species triad D. ambigua, D. tristis and D. obscura, in the genomic DNA of D. sinobscura and D. hubeiensis put the two sibling species in their close neighbourhood.     

Homologous recombination in Agrobacterium: potential implications for the genomic species concept in bacteria

According to current taxonomical rules, a bona fide bacterialspecies is a genomic species characterized by the genomic similarityof its members. It has been proposed that the genomic cohesionof such clusters may be related to sexual isolation, which limitsgene flow between too divergent bacteria. Homologous recombinationis one of the most studied mechanisms responsible for this geneticisolation. Previous studies on several bacterial models showedthat recombination frequencies decreased exponentially withincreasing DNA sequence divergence. In the present study, weinvestigated this relationship in the Agrobacterium tumefaciensspecies complex, which allowed us to focus on sequence divergencein the vicinity of the genetic boundaries of genomic species.We observed that the sensitivity of the recombination frequencyto DNA divergence fitted a log-linear function until approximately10% sequence divergence. The results clearly revealed that therewas no sharp drop in recombination frequencies at the pointwhere the sequence divergence distribution showed a "gap" delineatinggenomic species. The ratio of the recombination frequency inhomogamic conditions relative to this frequency in heterogamicconditions, that is, sexual isolation, was found to decreasefrom 8 between the most distant strains within a species to9 between the most closely related species, for respective increasesfrom 4.3% to 6.4% mismatches in the marker gene chvA. This meansthat there was only a 1.13-fold decrease in recombination frequenciesfor recombination events at both edges of the species border.Hence, from the findings of this investigation, we concludethat—at least in this taxon—sexual isolation basedon homologous recombination is likely not high enough to stronglyhamper gene flow between species as compared with gene flowbetween distantly related members of the same species. The 70%relative binding ratio cutoff used to define bacterial speciesis likely correlated to only minor declines in homologous recombinationfrequencies. Consequently, the sequence diversity, as a mechanisticfactor for the efficiency of recombination (as assayed in thelaboratory), appears to play little role in the genetic cohesionof bacterial species, and thus, the genomic species definitionfor prokaryotes is definitively not reconcilable with the biologicalspecies concept for eukaryotes.     

Molecular genetic characterization of the RD-114 gene family of endogenous feline retroviral sequences.

RD-114 is a replication-competent, xenotropic retrovirus which is homologous to a family of moderately repetitive DNA sequences present at ca. 20 copies in the normal cellular genome of domestic cats. To examine the extent and character of genomic divergence of the RD-114 gene family as well as to assess their positional association within the cat genome, we have prepared a series of molecular clones of endogenous RD-114 DNA segments from a genomic library of cat cellular DNA. Their restriction endonuclease maps were compared with each other as well as to that of the prototype-inducible RD-114 which was molecularly cloned from a chronically infected human cell line. The endogenous sequences analyzed were similar to each other in that they were colinear with RD-114 proviral DNA, were bounded by long terminal redundancies, and conserved many restriction sites in the gag and pol regions. However, the env regions of many of the sequences examined were substantially deleted. Several of the endogenous RD-114 genomes contained a novel envelope sequence which was unrelated to the env gene of the prototype RD-114 env gene but which, like RD-114 and endogenous feline leukemia virus provirus, was found only in species of the genus Felis, and not in other closely related Felidae genera. The endogenous RD-114 sequences each had a distinct cellular flank which indicates that these sequences are not tandem but dispersed nonspecifically throughout the genome. Southern analysis of cat cellular DNA confirmed the conclusions about conserved restriction sites in endogenous sequences and indicated that a single locus may be responsible for the production of the major inducible form of RD-114.     

Characterization and chromosomal distribution of a novel satellite DNA sequence of Japanese quail (Coturnix coturnix japonica)

A novel satellite DNA sequence of Japanese quail (Coturnix coturnix japonica) was isolated from genomic DNA digested with restriction endonuclease, Bg/II. Sequence analysis of three different-size clones revealed the presence of a tandem array of a GC-rich 41 bp repeated element. This sequence was localized by fluorescence in situ hybridization (FISH) primarily to microchromosomes of Japanese quail (2n = 78); approximately 50 of the 66 microchromosomes showed positive signals, although hybridization signals were also detected on chromosomes 4 and W. This satellite DNA did not cross-hybridize with genomic DNA of chicken (Gallus gallus) and Chinese painted quail (Excalfactoria chinensis) under moderately stringent conditions, suggesting that this class of repetitive DNA sequences was species specific and fairly divergent in Galliformes species.     

Concerted evolution of alpha satellite DNA: Evidence for species specificity and a general lack of sequence conservation among alphoid sequences of higher primates

We investigated relationships among alpha satellite DNA families in the human, gorilla, chimpanzee, and orangutan genomes by filter hybridization with cloned probes which correspond to chromosome-specific alpha satellite DNAs from at least 12 different human chromosomes. These include representatives of both the dimer-based and pentamer-based subfamilies, the two major subfamilies of human alpha satellite. In addition, we evaluated several high-copy dimer-based probes isolated from gorilla genomic DNA. Under low stringency conditions, all human probes tested hybridized extensively with gorilla and chimpanzee alpha satellite sequences. However, only pentameric and other non-dimeric human alphoid probes hybridized with orangutan alpha satellite sequences; probes belonging to the dimer subfamily did not cross-hybridize detectably with orangutan DNA. Moreover, under high stringency conditions, each of the human probes hybridized extensively only with human genomic DNA; none of the probes cross-hybridized effectively with other primate DNAs. Dimer-based gorilla alpha satellite probes hybridized with human and chimpanzee, but not orangutan, sequences under low stringency hybridization conditions, yet were specific for gorilla DNA under high stringency conditions. These results indicate that the alpha satellite DNA family has evolved in a concerted manner, such that considerable sequence divergence is now evident among the alphoid sequences of closely related primate species.     

Patchwork structure of a bovine satellite DNA

According to a previous restriction nuclease analysis, bovine 1.706 satellite DNA (density 1.706 g/cm3 in CsCl) is organized in an unusual structure of superimposed long- and short-range repeats (Streeck and Zachau, 1978). We have now determined the nucleotide sequence of this satellite DNA in both cloned fragments and fragments from the total satellite DNA. Each long-range repeat unit (about 2350 bp) is divided into four segments. Each segment consists of different variants of a basic 23 bp sequence which is itself composed of a dodecanucleotide and a related undecanucleotide. A total of 2400 nucleotides have been sequenced. Detailed analysis of the sequence divergence reveals that both the overall extent of divergence and the frequency of base changes at individual positions of the 23 bp repeats are characteristically different in the various segments. Preferentially methylated sites and a high incidence of symmetry elements are found. In two of the four segments, 22 of 23 bp of the prototype sequence are included in six overlapping elements of dyad symmetry and in a palindrome. A scheme for the evolution of the satellite DNA from a basic dodecanucleotide is proposed which is based on the different degrees of divergence for the various repeats superimposed in this satellite DNA.