Wing shape heritability and morphological divergence of the sibling species Drosophila mercatorum and Drosophila paranaensis

The fruit-flies Drosophila paranaensis and Drosophila mercatorum pararepleta are sibling species belonging to the repleta group. Females of these two species are normally considered to be morphologically indistinguishable while males only differ consistently in the morphology of their genitalia. These species are sympatric throughout a large area of their geographic distribution. In this study, we investigated the degree of morphological divergence between D. paranaensis and D. mercatorum pararepleta based on morphometric analysis of their wings. The ellipse method was used to describe the placement of the longitudinal and transversal wing veins as well as the size of the wing and the shape of its outline. The heritability under laboratory and field conditions was also estimated from the parameters generated. Multivariate analysis showed that wing morphology possessed sufficient differences to discriminate between the two species with a successful classification rate of 95-98% for females and 82-87% for males. The results of the autoclassification were confirmed by a cross-validation test for females (92-96%). Most measurements possessed significant natural heritability (a mean of 0.48 for D. mercatorum and 0.88 for D. paranaensis), indicating that the variation observed was related to differences in genes acting additively. The principal difference between the two species was in the placement of the posterior transverse wing vein. However, the pattern of morphological variation in the wings of both species was similar, possibly because of shared restrictions in wing development pathways.     

Localization of ecs, dor and swl genes in eight Drosophila species]

The DNA sequences from Drosophila melanogaster early ecdysterone-inducible puff 2B have been located in 8 Drosophila species by in situ hybridization. The location site of the ecs, dor and swi genes in D. funebris, D. virilis, D. hydei, D. repleta, D. mercatorum, D. paranaensis is a puff on the telomeric and of X chromosome; in D. kanekoi it is the puff in distal part of X chromosome; and in D. pseudoobscura it is the puff in proximal portion of X chromosome. So, conservative organization of DNA sequences located in D. melanogaster 2B puff could be suggested. Dispersed distribution of some DNA segments from the region studied in D. hydei chromosomes was revealed.     

The Molecular through Ecological Genetics of Abnormal Abdomen. II. Ribosomal DNA Polymorphism Is Associated with the Abnormal Abdomen Syndrome in DROSOPHILA MERCATORUM

Restriction endonuclease cleavage analyses of cloned and genomic DNA samples indicate that the structure of the DNA encoding the large cytoplasmic RNAs (rDNAs) is altered in Drosophila mercatorum lines which exhibit an abnormal abdomen (aa) phenotype. In a majority of the rDNA repeat units from aa flies, the 28S coding sequence is interrupted by a large [5-6 kilobase pairs (kbp)] insert. A subclone containing this inserted DNA (ins 3) hybridizes primarily to rDNA-containing sequences in in situ and genomic blot hybridization experiments. Additionally, genomic nitrocellulose blot hybridization analyses show that ins- containing rDNA repeat units are clustered in a spontaneously arising aa mutant. This rDNA alteration in D. mercatorum flies with the aa phenotype more closely resembles the bobbed (bb) defect of D. hydei than the bb defect of D. melanogaster, which involves alterations in rDNA copy number. By analogy with the other Drosophila systems, we propose that the altered D. mercatorum rDNA repeat units are defective in rRNA production at a critical stage. The lowered levels of rRNA ultimately would limit the concentration of ribosomes needed to produce large quantities of a protein (in these cases, juvenile hormone esterase) needed for normal development.     

Patterns of Variation in the Rdna Cistron within and among World Populations of a Mosquito, Aedes Albopictus (Skuse)

A restriction map was constructed of the ribosomal cistron in a mosquito, Aedes albopictus (Skuse). The 18s, 28s and nontranscribed spacer (NTS) regions were subcloned and used to probe for intraspecific variation. Seventeen populations were examined throughout the world range of the species. No variation was detected in the coding regions but extensive and continuous variation existed in the NTS. The NTS consisted of two nonhomologous regions. The first region contained multiple 190-bp AluI repeats nested within larger XhoI repeats of various sizes. There was a large number of length variants in the AluI repeat region of the NTS. No repeats were found in the second region and it gave rise to relatively fewer variants. An analysis of NTS diversity in individual mosquitoes indicated that most of the diversity arose at the population level. Discriminant analysis was performed on spacer types in individual mosquitoes and demonstrated that individuals within a population carried a unique set of spacers. In contrast with studies of the NTS in Drosophila populations, there seems to be little conservation of spacers in a population. The importance of molecular drive relative to drift and selection in the generation of local population differentiation is discussed.     

5S rDNA genome regions of Lens species.

The length variability of the nontranscribed spacer (NTS) of the 5S rDNA repeats was analyzed in species of the genus Lens by means of PCR amplification. The NTS ranged from approximately 227 to approximately 952 bp. The polymorphism detected was higher than previous NTS polymorphisms described in this genus. Three NTS length variants from Lens culinaris subsp. culinaris and 2 from Lens culinaris subsp. orientalis were sequenced. The culinaris NTS fragment lengths were 239, 371, and 838 bp, whereas the orientalis ones were 472 bp and 506 bp, respectively. As a result of sequence similarities, 2 families of sequences were distinguished, 1 including the sequences of 838 and 506 bp, and others with the sequences of 239, 371, and 472 bp. The 1st family was characterized by the presence of a repeated sequence designated A, whereas the 2nd family showed a single A sequence and other repeated sequences designated B, C, and D. The presence of an (AT)n microsatellite was also observed in the 2nd family of sequences. The fragments, which included the 239-bp and 838-bp NTS sequences, as well as the intergenic spacer (IGS) of the 18S-5.8S-26S ribosomal DNA also from L. culinaris subsp. culinaris, were used to localize the nucleolar organizer region (NOR) and the 5S rDNA loci in the chromosomes of several species of the genus Lens by means of fluorescence in situ hybridization (FISH). The selective hybridization of the 2 NTS probes allowed us to distinguish between different 5S rDNA chromosomal loci.     

Intraindividual and Interspecies Variation in the 5S rDNA of Coregonid Fish

This study was designed to characterize further the nontranscribed intergenic spacers (NTSs) of the 5S rRNA genes of fish and evaluate this marker as a tool for comparative studies. Two members of the closely related North American Great Lakes cisco species complex (Coregonus artedi and C. zenithicus) were chosen for comparison. Fluorescence in situ hybridization found the ciscoes to have a single multicopy 5S locus located in a C band-positive region of the largest submetacentric chromosome. The entire NTS was amplified from the two species by polymerase chain reaction with oligonucleotide primers anchored in the conserved 5S coding region. Complete sequences were determined for 25 clones from four individuals representing two discrete NTS length variants. Sequence analysis found the length variants to result from presence of a 130-bp direct repeat. No two sequences from a single fish were identical. Examination of sequence from the coding region revealed two types of 5S genes in addition to pseudogenes. This suggests the presence of both somatic and germline (oocyte) forms of the 5S gene in the genome of Coregonus. The amount of variation present among NTS sequences indicates that accumulation of variation (mutation) is greater in this multicopy gene than is gene conversion (homogenization). The high level of sequence variation makes the 5S NTS an inappropriate DNA sequence for comparisons of closely related taxa. Received: 22 August 1997 / Accepted: 31 October 1997     

Natural selection and ribosomal DNA in Drosophila

Natural populations of Drosophila mercatorum are variable for the number of X-linked 28S ribosomal genes bearing a 5-kilobase insert. A separate polymorphic X-linked gene controls whether 28S repeats bearing the insert are preferentially underreplicated during the formation of polytene tissue. Female flies having at least a third of their 28S genes bearing the insert and lacking the ability to preferentially underreplicate inserted repeats display the abnormal abdomen syndrome. The syndrome is characterized by retention of juvenile abdominal cuticle into the adult, a slowdown in larval developmental time, and an increase in early female fecundity. The life history traits are expressed in nature and provide a basis for strong natural selection. The abnormal abdomen syndrome should be favored whenever the adult age structure is skewed towards young individuals, and field studies confirm this prediction. The closely related species, Drosophila hydei, also bears these inserts and appears to be subject to similar selection. However, D. mercatorum responds to this selection primarily through the allelic variation that controls preferential underreplication, whereas D. hydei responds primarily through adjustment of the proportion of inserted 28S genes. This is interpreted to mean that the evolution of a multigene family arises from the interaction of population-level and DNA-level processes.     

Population Genetics of Tandem Repeats in Centromeric Heterochromatin: Unequal Crossing over and Chromosomal Divergence at the Responder Locus of Drosophila Melanogaster

The Responder (Rsp) locus in Drosophila melanogaster is the target locus of segregation distortion and is known to be comprised of a tandem array of 120-bp repetitive sequences. In this study, we first determined the large scale molecular structure of the Rsp locus, which extends over a region of 600 kb on the standard sensitive (cn bw) chromosome. Within the region, small Rsp repeat arrays are interspersed with non-Rsp sequences and account for 10-20% of the total sequences. We isolated and sequenced 32 Rsp clones from three different chromosomes. The main results are: (1) Rsp repeats isolated from the same chromosome are not more similar than those from different chromosomes. This implies either that there are more homologous exchanges at the Rsp locus than expected or, alternatively, that the second chromosomes of D. melanogaster have diverged from one another more recently at the centromeric heterochromatin than at the nearby euchromatin. (2) The repeats usually have a dimeric structure with an average difference of 16% between the left and right halves. The differences allow us to easily identify the products of unequal exchanges. Despite the large differences between the two halves, exchanges have occurred frequently and the majority of them fall within a 29-bp interval of identity between the two halves. Our data thus support the suggestion that recombination depends on short stretches of complete identity rather than long stretches of general homology. (3) Frequent unequal crossover events obscure the phylogenetic relationships between repeats; therefore, different parts of any single repeat could often have different phylogenetic histories. The high rate of unequal crossing over may also help explain the evolutionary dynamics of the Rsp locus.     

The phylogenetic relationships of flies in the family Drosophilidae deduced from mtDNA sequences.

The phylogenetic relationships of several taxa from representative genera, subgenera, groups, and subgroups in the Drosophilidae were examined using sequences from a 905-bp mtDNA fragment. Conventional cloning and sequencing techniques were used to obtain nucleotide sequences. In addition, polymerase chain reaction primers were designed for the rapid amplification and sequencing of this region for the species examined in the Drosophilidae. Phylogenetic analysis was done by cladistic techniques. Because of the coding nature of the 905-bp mtDNA fragment, several separate analyses of these sequences were performed. The genera Scaptomyza and Hirtodrosophila occupy ancestral branching positions in the molecular phylogeny. The genera Chymomyza and Zaprionus have intermediate branching positions, while the subgenera Drosophila and Sophophora are in the most derived position in the molecular phylogeny. Within the subgenus Sophophora, there is little resolution using these sequences, while within the subgenus Drosophila, D. melanica, D. funebris, and D. pinicola form a clade in a derived part of the phylogeny, with D. robusta and D. immigrans branching in an intermediate position in the phylogeny. D. mercatorum, a member of the repleta species group, occupies an ancestral position in the molecular phylogeny.     

The maintenance of polymorphism owing to differences in developmental time and competition

The effect of developmental time on the maintenance of an enzyme polymorphism is analyzed under the assumption that competition exists for a reproductively essential resource. Specifically, in laboratory vials Drosophila compete for oviposition and pupation sites. This system of competition serves as a prototype for the model with late-eclosing individuals being excluded from the reproductive pool. Two mating behaviors are modelled: (i) mating is with individuals from only the same cohort group and occurs only once, and (ii) mating is with all individuals that have eclosed up to that time and occurs daily. Numerical analyses are done on both mating structures using data from Drosophila mercatorum. The simulations show that mating structure and initial conditions do not affect the equilibrium genotype frequencies, but that slight changes in the level of competition can dramatically alter the equilibrium values.     

Evolutionary implications of developmental instability in parthenogenetic drosophila mercatorum. I. Comparison of several strains with different genotypes

Natural populations of sexually reproducing Drosophila mercatorum are capable of a very low rate of parthenogenesis, but this mode of reproduction has apparently never characterized an entirely asexual population in this species. The high abortion rate observed in laboratory parthenogenetic lines suggests that developmental constraints may cause the failure of this trait to spread in nature. To investigate the basis of this developmental instability and how it may affect the evolution of parthenogenesis in natural populations, early embryonic development was compared between one sexual and four parthenogenetic laboratory strains of D. mercatorum. There is a large amount of variation within a given parthenogenetic strain, suggesting that parthenogenesis is associated with a general breakdown of developmental stability. There is relatively little variation among different parthenogenetic strains, suggesting that most abortions are due to a feature inherent to parthenogenetic reproduction rather than a feature of a particular genome. Likewise, there is little variation between parthenogenetic and sexual strains in the causes of abortions, suggesting that the developmental problems encountered by parthenogenetic lineages are not unique to parthenogens. Thus, the failure of parthenogenesis to spread within D. mercatorum can be attributed to no particular developmental constraint per se operating after the initiation of embryogenesis. However, the overall increase in all developmental problems that occurs with the transition from sexual to parthenogenetic development suggests that the high degree of developmental instability associated with parthenogenesis may be considered a developmental constraint in its own right.     

Temporal and Spatial Heterogeneity of Mtdna Polymorphisms in Natural Populations of Drosophila mercatorum

Restriction endonuclease analysis of mtDNA was used to examine the genetic relatedness of several geographically separated isolines of the Drosophila mercatorum subgroup. In addition, we examined the temporal and spatial distribution of two mtDNA restriction site polymorphisms produced by the enzymes BstEII and BstNI at a single locality--Kamuela, Hawaii. Due to small sample sizes of some collections and the undesirable dependance of the estimation of polymorphism frequency on its variance, an arcsin square root transformation of the frequency data was used. We also use an Fst estimator of our transformed frequencies to demonstrate considerable spatial and temporal differentiation within the Kamuela population. In contrast, isozyme data from the same population reveals no pattern of differentiation. The temporal and geographic heterogeneity and population subdivision detected with mtDNA analysis also is consistent with the known dispersal behavior and ecological constraints of this species. The mtDNA data in conjunction with the isozyme data show that the population structure of the Kamuela D. mercatorum is close to the boundary line separating panmixia from subdivision, a conclusion that could not be made from isozyme data alone.     

Molecular population genetics of Drosophila subtelomeric DNA

DNA sequence surveys in yeast and humans suggest that the forces shaping telomeric polymorphism and divergence are distinctly more dynamic than those in the euchromatic, gene-rich regions of the chromosomes. However, the generality of this pattern across outbreeding, multicellular eukaryotes has not been determined. To characterize the structure and evolution of Drosophila telomeres, we collected and analyzed molecular population genetics data from the X chromosome subtelomere in 58 lines of North American Drosophila melanogaster and 29 lines of African D. melanogaster. We found that Drosophila subtelomeres exhibit high levels of both structural and substitutional polymorphism relative to linked euchromatic regions. We also observed strikingly different patterns of variation in the North American and African samples. Moreover, our analyses of the polymorphism data identify a localized hotspot of recombination in the most-distal portion of the X subtelomere. While the levels of polymorphism decline sharply and in parallel with rates of crossing over per physical length over the distal first euchromatic megabase pairs of the X chromosome, our data suggest that they rise again sharply in the subtelomeric region (approximately 80 kbp). These patterns of historical recombination and geographic differentiation indicate that, similar to yeast and humans, Drosophila subtelomeric DNA is evolving very differently from euchromatic DNA.     

Life-history changes that accompany the transition from sexual to parthenogenetic reproduction in Drosophila mercatorum

In spite of the predicted genetic and ecological costs of sex, most natural populations maintain sexual reproduction, even those capable of facultative parthenogenesis. Unfertilized eggs from natural populations of Drosophila mercatorum occasionally develop into viable adults, but obligately parthenogenetic populations are unknown in this species. To evaluate the microevolutionary forces that both favor and constrain the evolution of parthenogenesis in D. mercatorum, we have measured parthenogenetic rates across a natural, sexually reproducing population and characterized the life-history changes that accompany the transition from sexual to parthenogenetic reproduction in laboratory strains. A highly significant difference in parthenogenetic rate was found between two populations in close geographic proximity, with increased rate found with lower population density. Laboratory strains of parthenogenetic females suffered increased mortality and reduced egg viability relative to their virgin counterparts from a sexual strain. Lifetime egg production was similar across all strains, but a shift in peak egg production to an earlier age also occurred. The combination of these life-history traits resulted in a higher net reproductive value for sexual females, but because they also had a longer generation time, intrinsic rate of increase was not as dramatically different from parthenogenetic females. In environments with high early mortality, there may be no fitness disadvantage to parthenogenesis, but the predicted ecological advantage of a twofold increase in intrinsic rate of increase was not realized. These results support the theory of Stalker (1956) that parthenogenesis is favored in environments in which sexual reproduction is difficult or impossible.     

Structural analysis of glycerol-3-phosphate dehydrogenase from severalDrosophila species

This report describes preliminary protein structural studies of glycerol-3-phosphate dehydrogenase (alpha-GPDH) from Drosophila spp. and an important innovative feature of our enzyme purification protocol. The scheme involves the coupling of substrate (alpha-glycerophosphate) elution from CM-Sephadex and cofactor (NADH) elution from Affi-Gel blue resin. Using this method a 32.7% yield and a 111-fold purification were obtained from a D. melanogaster line carrying the alpha-GpdhS allele at the alpha-Gpdh locus. The product obtained from 0 to 3-day-old adult flies was electrophoretically homogeneous and consisted mainly of the adult alpha-GPDH-1 isozyme. The method was used to obtain alpha-GPDH protein from D. melanogaster (two lines), D. hydei, D. immigrans, and D. mercatorum. Peptide mapping revealed structural differences among the enzymes from the different species, and amino acid sequencing showed many similarities between D. melanogaster alpha-GPDH and the rabbit muscle enzyme.     

The Molecular through Ecological Genetics of Abnormal Abdomen. III. Tissue-Specific Differential Replication of Ribosomal Genes Modulates the Abnormal Abdomen Phenotype in DROSOPHILA MERCATORUM

The abnormal abdomen (aa) syndrome in Drosophila mercatorum is controlled by two major X-linked genetic elements. We have previously shown that the major X-linked element of aa is associated with the presence of large inserts in the 28S gene of the ribosomal RNA (rDNA) genes. We show that, in polytene tissue of wild-type D. mercatorum, the uninterrupted rDNA repeats are overreplicated relative to interrupted repeats. Uninterrupted rDNA repeats are also overreplicated in polytene tissue of hybrid larval offspring from wild-type and aa parents. This overreplication of uninterrupted repeats is not observed in diploid tissues of wild-type hybrids (of wild-type and aa parents) and homozygous aa larvae or in polytene tissue of aa larvae. Furthermore, molecular analysis of an aa line that has reverted to the wild type indicates that the reversion phenomenon is associated with the ability to overreplicate uninterrupted rDNA repeats in polytene tissues. The patterns of differential replication of rDNA genes in wild-type hybrids and aa larvae of D. mercatorum offer a possible mechanism for the tissue-specific control of the aa phenotype and suggest that the molecular basis for the second X-linked genetic element of aa is involved in the control of differential replication in polytene tissues.     

Molecular genetic variation in the centromeric region of the X chromosome in three Drosophila ananassae populations. II. The Om(1D) locus

Restriction-site and sequence-length polymorphism in the Om(1D) locus region on the X chromosome in Drosophila ananassae was investigated for three natural populations (from Burma, India, and Brazil), by using hexanucleotide-recognizing restriction enzymes. The estimates of average heterozygosity per nucleotide (pi) were 0.0085, 0.0043, and 0.0004 for the Burma, India, and Brazil populations, respectively, and the average frequencies of insertions/deletions were 0.078, 0.054, and 0.007/chromosome/kb. While the pi values at this locus are similar to the estimates obtained from other euchromatic loci in D. ananassae or in other Drosophila species, the frequencies of insertions/deletions are much higher than those previously reported from Drosophila. The exceptionally high frequencies of length polymorphisms in the Burmese sample and, to a lesser extent, in the Indian sample indicate that the hypermutability of Om(1D), caused by the frequent insertion of the transposable element tom, may be due to locus-specific rather than to tom element-specific properties. The low level of nucleotide variation in the Brazilian population seems to be due to a recent bottleneck of population size. This population was apparently founded in recent years by a small number of individuals and has been relatively isolated ever since.     

Dosage Compensation Regulatory Proteins and the Evolution of Sex Chromosomes in Drosophila

In the fruitfly Drosophila melanogaster, the four male-specific lethal (msl) genes are required to achieve dosage compensation of the male X chromosome. The MSL proteins are thought to interact with cis-acting sites that confer dosage compensation to nearby genes, as they are detected at hundreds of discrete sites along the length of the polytene X chromosome in males but not in females. The histone H4 acetylated isoform, H4Ac16, colocalizes with the MSL proteins at a majority of sites on the D. melanogaster X chromosome. Using polytene chromosome immunostaining of other species from the genus Drosophila, we found that X chromosome association of MSL proteins and H4Ac16 is conserved despite differences in the sex chromosome karyotype between species. Our results support a model in which cis-acting regulatory sites for dosage compensation evolve on a neo-X chromosome arm in response to the degeneration of its former homologue.     

Nucleotide sequence variability in the nontranscribed spacer of the rRNA locus in the oyster parasite Perkinsus marinus.

We examined the sequence variability of the nontranscribed spacer (NTS) and internal-transcribed spacer (ITS1 and ITS2) domains of the rRNA locus of Perkinsus marinus from Maryland, Florida, and Louisiana. The sequence of P. marinus DNA including the 5S rRNA, NTS, small subunit (SSU) rRNA, ITSI, and ITS2 regions confirmed their contiguity in the rRNA locus and revealed differences at 28 positions with the SSU rRNA sequences published earlier. The 307-bp polymerase chain reaction (PCR)-amplified fragments from the NTS domain of the various P. marinus isolates revealed the presence of 2 distinct sequences, designated as types I and II, that differed at 6 defined nucleotide positions. Based on these differences, nested PCR and restriction enzyme digests were used to distinguish between the 2 types. Sequences of the ITS1 and ITS2 domains of samples from either NTS type I (n = 3) or type II (n = 3) showed no variation and were identical to published sequences. Frequencies of the P. marinus NTS sequence types I and II in infected oysters varied with the geographic origin of the samples. All Maryland samples examined (n = 19) corresponded to the NTS type I sequence, the type II was the most frequent in the Florida samples (n = 17), and both types were about equally represented in the Louisiana samples (n = 19), with both sequence types found in individual oyster specimens. Although it has been suggested that P. marinus is diploid, it remains to be determined if both NTS sequence types can be present in a single P. marinus trophozoite.