Nontranscribed spacers in Drosophila ribosomal DNA

Ribosomal DNA nontranscribed spacers in Drosophila virilis DNA have been examined in some detail by restriction site analysis of cloned segments of rDNA, nucleic acid hybridizations involving unfractionated rDNA, and base composition estimates. The overall G+C content of the spacer is 27–28%; this compares with 39% for rDNA as a whole, 40% for main band DNA, and 26% for the D. virilis satellites. Much of the spacer is comprised of 0.25 kb repeats revealed by digestion with Msp I, Fnu DII or Rsd I, which terminate very near the beginning of the template for the ribosomal RNA precursor. The spacers are heterogeneous in length among rDNA repeats, and this is largely accounted for by variation among rDNA units in the number of 0.25 kb elements per spacer. Despite its high A+T content and the repetitive nature of much of the spacer, and the proximity of rDNA and heterochromatin in Drosophila, pyrimidine tract analysis gave no indication of relatedness between the spacer and satellite DNA sequences. Species of Drosophila closely related to D. virilis have rDNA spacers that are homologous with those in D. virilis to the extent that hybridization of a cloned spacer segment of D. virilis rDNA to various DNA is comparable with hybridization to homologous DNA, and distributions of restriction enzyme cleavage sites are very similar (but not identical) among spacers of the various species. There is spacer length heterogeneity in the rDNA of all species, and each species has a unique major rDNA spacer length. Judging from Southern blot hybridization, D. hydei rDNA spacers have 20–30% sequence homology with D. virilis rDNA spacers, and a repetitive component is similarly sensitive to Msp I and Fnu DII digestion, D. melanogaster rDNA spacers have little or no homology with counterparts in D. virilis rDNA, despite a similar content of 0.25 kb repetitive elements. In contrast, sequences in rDNA that encode 18S and 28S ribosomal RNA have been highly conserved during the divergence of Drosophila species; this is inferred from interspecific hybridizations involving ribosomal RNA and a comparison of distributions of restriction enzyme cleavage sites in rDNA.Dedicated to Professor Wolfgang Beermann on the occasion of his sixtieth birthday     

Nuclear DNA divergence among Lathyrus species

Among diploid Lathyrus species a threefold variation in nuclear DNA amount is attributable to differences in the amount of repetitive DNA. Cross reassociation among repetitive and among non-repetitive DNA fractions from different species shows substantial divergence in DNA composition. The divergence in base composition is correlated with nuclear DNA amount. The degree of divergence is of the same order of magnitude in both the repetitive and nonrepetitive fractions.     

Changes in DNA composition in the evolution of Vicia species

Summary The composition of nuclear DNA in 3 Vicia species are compared. The species V. eriocarpa, V. johannis and V. melanops are from three separate subgeneric sections of Vicia and show a fourfold variation in their amounts of nuclear DNA. DNA melting experiments, buoyant density gradient analysis and Cot reassociation experiments show that the quantitiative change in nuclear DNA between the three species is achieved by changes in the amounts of both repetitive and nonrepetitive DNA sequences. It is suggested that while the increase in the repetitive fraction is achieved by the proliferation of repetitive base sequences the increase in the nonrepetitive fraction is due to the steady accretion of highly diverged base sequences resulting from mutations, deletions, insertions and base sequence rearrangements among families of repetitive sequences.     

Characteristics of nuclear DNA in the genus Oryza

Summary DNAs have been isolated from various Oryza species and studied using physical techniques. The percent of guanine plus cytosine has been determined by thermal denaturation. While the base composition varied between the species, no heterogeneity in the base pair distribution was observed. Renaturation kinetics data of DNAs from different species show that the proportion of repeated DNA sequences vary considerably depending on the DNA content per cell, whereas the nonrepetitive DNA component remains relatively constant. These results suggest that in addition to a small range of DNA variation between the species, changes in the base composition and proportion of repeated sequences have accompanied divergence of the species within the genus.     

SEQUENTIAL RADIATIONS AND PATTERNS OF SPECIATION IN THE HAWAIIAN CRICKET GENUS LAUPALA INFERRED FROM DNA SEQUENCES

The tremendous diversity of endemic Hawaiian crickets is thought to have originated primarily through intraisland radiations, in contrast to an interisland mode of diversification in the native Hawaiian Drosophila. The Hawaiian cricket genus Laupala (family Gryllidae) is one of several native genera of flightless crickets found in rain-forest habitat across the Hawaiian archipelago. I examined the phylogenetic relationships among mitochondrial DNA (mtDNA) sequences sampled from 17 species of Laupala, including the 12S ribosomal RNA (rRNA), transfer RNA (RNA)^val and 16S rRNA regions. The distribution of mtDNA variants suggests that species within Laupala are endemic to single islands. The phylogenetic estimate produced from both maximum likelihood and maximum parsimony supports the hypothesis that speciation in Laupala occurred mainly within islands. The inferred biogeographical history suggests that diversification in Laupala began on Kauai, the oldest rain-forested Hawaiian island. Subsequently, colonization to younger islands in the archipelago resulted in a radiation of considerable phylogenetic diversity. Phylogenetic patterns in mtDNA are not congruent with prior systematic or taxonomic hypotheses. Hypotheses that may explain the conflict between the phylogenetic patterns of mtDNA variation and the species taxonomy are discussed.     

Evolution of Y chromosomal lampbrush loop DNA sequences of Drosophila

The evolutionary conservation of Y chromosomal DNA sequences of Drosophila hydei in different species of the genus Drosophila was studied by in situ hybridization and on genomic DNA blots of restriction enzyme digested DNA. We demonstrated that Y specific DNA sequences, which form major parts of lampbrush loops related to the male fertility genes, are only retained in a few closely related species during evolution. Other Y chromosomal DNA sequences, also present in lampbrush loops but with homology to autosomal and X chromosomal locations, were found in distant species. We propose a model for the evolution of the Y chromosomal lampbrush loops.     

FOUNDER EFFECTS AND THE RATE OF MITOCHONDRIAL DNA EVOLUTION IN HAWAIIAN DROSOPHILA

The nearly-neutral-mutation theory predicts that populations with small effective sizes will undergo more rapid molecular evolution than populations with very large effective sizes. In particular, Ohta (1976) predicted that populations of Hawaiian Drosophila that are known to have small population sizes and to experience repeated population bottlenecks due to founder events should show a more rapid rate of molecular evolution relative to the rate of molecular evolution of species with large population sizes such as the continental Drosophila. In this paper we test this prediction by comparing the rate of molecular evolution in two closely related lineages of Hawaiian Drosophila that have experienced very different evolutionary histories. Both lineages belong to the planitibia subgroup of Hawaiian Drosophila. The beta lineage (which includes D. silvestris, D. planitibia, D. differens, and D. hemipeza) has undergone repeated founder events, as evidenced by their geographic distribution and behavioral biology. On the other hand, evidence on geographic distribution and behavior indicates that the alpha lineage (which includes D. melanocephala, D. cyrtaloma, and D. neoperkinsi) has arisen from large ancestral populations without founder effects. The mitochondrial DNA data reveal that, within a lineage, the rate of molecular evolution is rather uniform, while all comparisons between the two lineages show that the rate of molecular evolution in the beta lineage is three times that of the alpha lineage. This analysis strongly supports the predictions made by Ohta.     

Evolutionary distances in hawaiian drosophila measured by DNA reassociation

Summary Comparisons of the sequence divergence of three species of Hawaiian Drosophila have been made by hybridization of single-copy tracer DNA of each of the species with driver DNA from each species, and measurement of the average melting temperature (Tma) in a chaotropic solvent (2.4 M tetraethylammonium chloride) which minimizes differences due to base composition. Correction was made for the length of hybrid duplex regions to obtain the reduction in thermal stability due to divergence.An accuracy of ± 0.2°C was achieved and the mean reduction in Tm for hybridization betweenD. heteroneura andD. silvestris (found only on the island of Hawaii) was 0.55°C and betweenD. picticornis, found only on the island of Kauai, and the other two species was 2.13°C. The rate of DNA change is estimated to be between 0.2 and 0.4%/My by assuming that theD. heteroneura-D. silvestris divergence occurred 0.8 My ago and the divergence between these species andD. picticornis between 4 and 6 My ago.The general single copy DNA sequence divergence appears to be very much greater than the minimal coding region sequence divergence previously estimated from allozyme studies.     

Analysis of a dispersed repetitive DNA sequence in isogenic lines of Drosophila

The location of sequences homologous to a cloned D. melanogaster DNA segment, Dm 25, has been examined in polytene chromosomes by hybridization in situ. Dm 25 localizes to multiple sites and shows variation in patterns between different strains and among individuals within wild-type laboratory strains. Analysis of numerous geographically distinct isogenic lines suggests that Dm 25 patterns are determined by germ-line factors and are not the product of strictly somatic events. In general there is wide variation in Dm 25 patterns among different lines, but a significant number of sites are common to two or more distinct lines. Hybridization to restriction digests of genomic DNA suggests that Dm 25 is a moderately repetitive, conserved sequence whose copies are dispersed throughout the genome. Analysis of species other than melanogaster indicates a significant divergence in structure of sequences homologous to Dm 25 as well as a drastic reduction in amount of homology to the melanogaster sequence.     

DNA sequences repaired at pachytene exhibit strong homology among distantly related higher plants

Moderately repetitive DNA sequences in Lilium (cv Enchantment) which undergo a meiotic-specific repair synthesis during pachytene (P-DNA) were previously shown to exist as families of very low internal sequence divergence. The present study concerns P-DNA sequence preservation among higher plants. The relative abundance of these sequences in a variety of plant species and their divergence relative to Enchantment P-DNA was determined through C₀t analysis and thermal denaturation of hybrid duplexes. Nearly all of the P-DNA sequence families of Enchantment were found to be present in the genomes of a number of monocot species and the dicot Vicia faba. Sequence content is highly conserved, with less than 6% divergence between Lilium and distantly related species such as Zea mays and Secale cereale. However, the number of repeats per P-DNA family varies considerably in different species, being particularly low among the Poales. P-DNA differs from most high thermal stability (HTS) sequence families of Enchantment which, although exhibiting a high degree of internal homology, are not present as repetitive DNA in the genomes of the other species examined. For most HTS families, the lack of internal divergence probably reflects their fairly recent introduction into the moderately repetitive DNA class, while P-DNA sequences represent evolutionarily ancient families which are the products of strong selective pressure for an indispensable meiotic function.     

The DNA sequences of cloned complex satellite DNAs from Hawaiian Drosophila and their bearing on satellite DNA sequence conservation

A class of restriction endonuclease fragments near 185 bp in length and comprising approximately 20% of the genomes of 3 species of Hawaiian Drosophila has been cloned using bacteriophage M13. The nucleotide sequences of 14 clones have been determined and the variation between clones has been found to be due to deletions and base changes. Analyses of uncloned material show that the cloning system itself does not introduce the variation. The variation of the basic repeat within and between species is high; 15% due to deletions and 10% due to base changes. The Drosophila data are similar in many respects to both the 23 bp calf satellite results (Pech et al., 1979b) and those from sequence analyses of the 170 bp primate restriction fragments (Rubin et al, 1979; Donehower et al., 1980, Wu and Manuelidis, 1980). The intraspecies level of base changes and deletions in the calf satellite approaches 25% as does that in the human/African green monkey/baboon comparisons. The between species variation in the primate group is near 35%. Direct sequencing methods thus reveal a widespread sequence heterogeneity in both invertebrate and mammalian satellite systems of long or short repeat length. This heterogeneity does not support the strict sequence conservation implied by the library hypothesis, which claims a functional role in speciation for the rigid conservation of satellite DNA sequences (Fry and Salser, 1977). Furthermore the Drosophila and primate data reveal that satellite DNAs can change rapidly, though nonrandomly, at the nucleotide sequence level in a relatively closely knit group such as the Hawaiian species, as well as in more distantly related species from amongst the primates. We draw two major conclusions. There is no universal attribute of satellite DNA sequence per se, the only biological variable to date being the amount of satellite DNA and its effects in the germ line. Many aspects of satellite DNA evolution conform to Kimura's (1979) concepts of neutrality.     

Unequal evolutionary rates in the <Emphasis Type="Italic">Drosophila virilis</Emphasis> species group: I. The use of phylogeny-based Takezaki’s tests

The aim of this study is to test the hypothesis about an inconsistency in time estimation of the divergence and topology of species Drosophila kanekoi, D. ezoana, and D. littoralis which is caused by the irregular accumulation rate of replacements in different DNA sequences used for analysis and in evolutionary lineages. The phylogenetic relationships based on sequences of five genes among 11 Drosophila species of the virilis group are revised, and estimation of molecular clock regularity in several phylogenetic lineages of this group is given. It was shown that Drosophila kanekoi, D. ezoana, and D. littoralis contituted in a single cluster, which was most related to the subphylad montana. The irregularity of molecular clocks was shown for the highly conservative sequences of the mt 12S-16S rRNA and Ras1 genes, the replacements of which were predominantly neutral. The irregularity of the molecular clock between several phylads and phylogenetic trees of subphylads lummei and kanekoi was revealed.     

CHEMICAL COMMUNICATION IN HAWAIIAN DROSOPHILA

We are interested in elucidating the extent to which lekking Hawaiian Drosophila species have diverged from their continental counterparts, which engage in sexual behavior at communal food sources, with regard to the chemical communication systems that the flies employ. Accordingly, we have analyzed flies from three closely related Hawaiian Drosophila species in the adiastola subgroup. These species are of interest because the males engage in a unique behavior: while courting, they raise their abdomens over their heads and emit anal droplets. Analysis of the flies' behavior, the hydrocarbons in males' anal droplets, and males' cuticular hydrocarbons suggest that females' responses to males may be mediated by cuticular pheromones and/or pheromones in males' extruded droplets that enable the females to distinguish conspecific from heterospeciflc males. Conversely, perception of cuticular hydrocarbons from conspecific females enables D. adiastola males to distinguish females from a closely related species from conspecific females. On the basis of these observations, we suggest that the adiastola subgroup species are unique among drosophilids in that they utilize an anal droplet-mediated pheromone communication system, some or all components of which are species specific. However, the lekking Hawaiian Drosophila species are similar to D. melanogaster and related continental species in that the Hawaiian flies employ a cuticular pheromone communication system, some components of which are sex and species-specific.     

Evolution of the autosomal chorion cluster inDrosophila. IV. The HawaiianDrosophila: Rapid protein evolution and constancy in the rate of DNA divergence

Summary Autosomal chorion geness18, s15, ands19 are shown to diverge at extremely rapid rates in closely related taxa of HawaiianDrosophila. Their nucleotide divergence rates are at least as fast as those of intergenic regions that are known to evolve more extensively between distantly related species. Their amino acid divergence rates are the fastest known to date. There are two nucleotide replacement substitutions for every synonymous one. The molecular basis for observed length and substitution mutations is analyzed. Length mutations are strongly associated with direct repeats in general, and with tandem repeats in particular, whereas the rate for an average transition is twice that for an average transversion.The DNA sequence of the cluster was used to construct a phylogenetic tree for five taxa of the Hawaiian picture-winged species group ofDrosophila. Assignment of observed base substitutions occurring in various branches of the tree reveals an excess of would-be homoplasies in a centrally localized 1.8-kb segment containing thes15 gene. This observation may be a reflection of ancestral excess polymorphisms in the segment. The chorion cluster appears to evolve at a constant rate regardless of whether the central 1.8-kb segment is included or not in the analysis. Assuming that the time of divergence ofDrosophila grimshawi and theplanitibia subgroup coincides with the emergence of the island of Kauai, the overall rate of base substitution in the cluster is estimated to be 0.8% million years, whereas synonymous sites are substituted at a rate of 1.2%/million years.     

Single copy DNA and structural gene sequence relationships among four sea urchin species

Measurements of the divergence of single copy DNA sequences among four sea urchin species are presented. At a standard criterion for reassociation (0.12 M phosphate buffer, 60° C, hydroxyapatite binding) we observe the following extents of reaction and reductions in thermal stability for single copy DNA reassociation between Strongylocentrotus purpuratus tracer and heterologous driver DNA: S. dröbachiensis 68% and 2.5°C; S. franciscanus 51% and 3.5° C; Lytechinus pictus 12% and 7.5° C. The implied extents of sequence relatedness are consistent with the phylogenetic relationships of these species. The rate of single copy sequence divergence in the evolutionary lines leading to the Strongylocentrotus species is estimated to be 0.06–0.35% per million years. The rate of divergence of total single copy sequence has been compared to that of structural gene sequences represented in S. purpuratus gastrula polysomal messenger RNA. When closely related species, S. purpuratus and S. franciscanus, are compared, these polysomal sequences are found to diverge at a lower rate than does the total single copy sequence. For two very distantly related species, S. purpuratus and L. pictus, a small fraction of the single copy DNA sequence is probably conserved. These conserved sequences are not enriched in their content of structural gene sequences.Also staff member, Carnegie Institution of Washington, Washington, D.C. 20015     

EVOLUTIONARY IMPLICATIONS OF DNA DIVERGENCE IN THE DROSOPHILA OBSCURA GROUP

Using DNA–DNA hybridization, we have determined the degree of single-copy DNA (scDNA) divergence among eight species of the Drosophila obscura group. These include Old World and New World species as well as members of two subgroups. Contrary to classical systematics, members of the affinis subgroup are more closely related to American members of the obscura subgroup than are Old World species. The Old World species are not a monophyletic group. The degree of scDNA divergence among species is not necessarily correlated with morphology, chromosomal divergence, or ability to form hybrids. A unique pattern of hybrid formation was found: species separated by a ΔT_m of 6.5°C can form hybrids whereas species separated by a ΔT_m of 2.5°C cannot. As with other groups of Drosophila, the obscura group has discrete parts of the genome evolving at very different rates. The slow evolving fraction of the nuclear genome is evolving at about the same rate as mitochondrial DNA. The additional scDNA divergence accompanying the step from partial reproductive isolation (between North American pseudoobscura and the isolated Bogotà population) to full isolation is very small. The resolution of the technique was challenged by five closely related taxa with a maximum ΔT_m of 2.5°C separating them; the taxa were unambiguously resolved and the “correct” phylogeny recovered. Finally, there is some indication that scDNA in the obscura group may be evolving considerably slower than in the melanogaster subgroup.     

Phylogenetic utility of the internal transcribed spacers of nuclear ribosomal DNA in plants: an example from the compositae.

The internal transcribed spacer (ITS) region of 18-26S nuclear ribosomal DNA was sequenced in 12 representatives of the Compositae subtribe Madiinae and two outgroup species to assess its utility for phylogeny reconstruction. High sequence alignability and minimal length variation among ITS 1, 5.8S, and ITS 2 sequences facilitated determination of positional homology of nucleotide sites. In pairwise comparisons among Madiinae DNAs, sequence divergence at unambiguously aligned sites ranged from 0.4 to 19.2% of nucleotides in ITS 1 and from 0 to 12.9% of nucleotides in ITS 2. Phylogenetic relationships among ITS sequences of Hawaiian silversword alliance species (Argyroxiphium, Dubautia, and Wilkesia) and California tarweed taxa in Adenothamnus, Madia, Raillardella, and Raillardiopsis are highly concordant with a chloroplast DNA-based phylogeny of this group. Maximally parsimonious trees from ITS and chloroplast DNA data all suggest (a) origin of the monophyletic Hawaiian silversword alliance from a California tarweed ancestor, (b) closer relationship of the Hawaiian species to Madia and Raillardiopsis than to Adenothamnus or Raillardella, (c) paraphyly of Raillardiopsis, a segregate of Raillardella, and (d) closer relationship of Raillardiopsis to Madia and the silversword alliance than to Raillardella. These findings indicate that the ITS region in plants should be further explored as a promising source of nuclear phylogenetic markers.     

Role of very slightly deleterious mutations in molecular evolution and polymorphism

Several models of multiple slightly deleterious alleles are reviewed and theoretical consequences of slightly negative selection are discussed in conjunction with evolution and variation at the molecular level. Facts are organized which may be satisfactorily explained by the hypothesis of very slightly deleterious mutations. They are: (1) There appears to be an upper limit in heterozygosity for protein loci as measured by electrophoresis. (2) The excess of rare alleles is more pronounced in Drosophila than in man. (3) Correlation of heterozygosities at a locus among sibling species of the Drosophila willistoni group is too high compared to what is expected by the strict neutral theory, while it is not so among human races and between man and chimpanzee. (4) The rate of protein divergence is exceptionally high in Hawaiian Drosophila.     

Single copy DNA homology in sea stars

Summary The sequence homology in the single copy DNA of sea stars has been measured. Labeled single copy DNA fromPisaster ochraceus was reannealed with excess genomic DNA fromP. brevispinus, Evasterias troschelii, Pycnopodia helianthoides, Solaster stimpsoni, andDermasterias imbricata. Reassociation reactions were performed under two criteria of salt and temperature. The extent of reassociation and thermal denaturation characteristics of hybrid single copy DNA molecules follow classical taxonomic lines.P. brevispinus DNA contains essentially all of the sequences present inP. ochraceus single copy tracer whileEvasterias andPycnopodia DNAs contain 52% and 46% of such sequences respectively. Reciprocal reassociation reactions with labeledEvasterias single copy DNA confirm the amount and fidelity of the sequence homology. There is a small definite reaction of uncertain homology betweenP. ochraceus single copy DNA andSolaster orDermasterias DNA. SimilarlySolaster DNA contains sequences homologous to approximately 18% ofDermasterias unique DNA. The thermal denaturation temperatures of heteroduplexes indicate that the generaPisaster andEvasterias diverged shortly after the divergence of the subfamilies Pycnopodiinae and Asteriinae. The twoPisaster species diverged more recently, probably in the most recent quarter of the interval since the separation of the generaPisaster andEvasterias.     

Evaluation of intra- and interspecific divergence of satellite DNA sequences by nucleotide frequency calculation and pairwise sequence comparison

Satellite DNA sequences are known to be highly variable and to have been subjected to concerted evolution that homogenizes member sequences within species. We have analyzed the mode of evolution of satellite DNA sequences in four fishes from the genusDiplodus by calculating the nucleotide frequency of the sequence array and the phylogenetic distances between member sequences. Calculation of nucleotide frequency and pairwise sequence comparison enabled us to characterize the divergence among member sequences in this satellite DNA family. The results suggest that the evolutionary rate of satellite DNA inD. bellottii is about two-fold greater than the average of the other three fishes, and that the sequence homogenization event occurred inD. puntazzo more recently than in the others. The procedures described here are effective to characterize mode of evolution of satellite DNA. Published: March 4, 2003