Inferring the evolutionary history of Drosophila americana and Drosophila novamexicana using a multilocus approach and the influence of chromosomal rearrangements in single gene analyses

The evolutionary history of closely related organisms can prove sometimes difficult to infer. Hybridization and incomplete lineage sorting are the main concerns; however, genome rearrangements can also influence the outcome of analyses based on nuclear sequences. In the present study, DNA sequences from 12 nuclear genes, for which the approximate chromosomal locations are known, have been used to estimate the evolutionary history of two forms of Drosophila americana ( Drosophila   americana americana and Drosophila americana texana ) and Drosophila novamexicana ( virilis group of species). The phylogenetic analysis of the combined data set resulted in a phylogeny showing reciprocal monophyly for D. novamexicana and D. americana . Single gene analyses, however, resulted in incongruent phylogenies influenced by chromosomal rearrangements. Genetic differentiation estimates indicated a significant differentiation between the two species for all genes. Within D. americana , however, there is no evidence for differentiation between the chromosomal forms except at genes located near the X/4 fusion and Xc inversion breakpoint. Thus, the specific status of D. americana and D. novamexicana is confirmed, but there is no overall evidence for genetic differentiation between D. a. americana and D. a. texana , not supporting a subspecific status. Based on levels of allele and nucleotide diversity found in the strains used, it is proposed that D. americana has had a stable, large population during the recent past while D. novamexicana has speciated from a peripheral southwestern population having had an ancestral small effective population size. The influence of chromosomal rearrangements in single gene analyses is also examined.     

Aggregation pheromones in five taxa of the Drosophila virilis species group

ABSTRACT. Aggregation pheromones have been demonstrated in the closely related taxa: Drosophila americana americana Spencer, D. a. texana Patterson, D. novamexicana Patterson, and D. lummei Hackman. These pheromones function much as has been reported previously for D. virilis Sturtevant. The compounds are produced by sexually mature males, but both sexes respond in a wind-tunnel olfactometer. In all species except D. lummei , a 21-carbon alkene is an important pheromone component. In D. virilis the hydrocarbon is (Z)-10-heneicosene (Z10–21), but in D. a. americana, D. a. texana and D. novamexicana it is (Z)-9-heneicosene (Z9-21). All these taxa respond best to the heneicosene which they produce. D. lummei possesses no heneicosenes but, curiously, responds well to both Z9-21 and Z10-21. All species possess five male-specific esters which were previously discovered in D. virilis : methyl tiglate, ethyl tiglate, isopropyl tiglate, methyl hexanoate and ethyl hexanoate. Ethyl tiglate is the most abundant in each case. Responses to the esters vary among the taxa, ranging from highly significant in D. lummei , particularly to ethyl tiglate, to not demonstrable in D. a. americana. Variability in ester response has also been demonstrated between two strains of D. virilis. In all cases the crude male-derived pheromone is synergistic with an extract of fermented willow bark, on which oviposition is said to occur.     

A multilocus microsatellite phylogeny of the Drosophila virilis group

We used a set of 48 polymorphic microsatellites derived from Drosophila virilis to infer phylogenetic relationships in the D. virilis clade. Consistent with previous studies, D. virilis and D. lummei were the most basal species of the group. Within the D. montana phylad, the phylogenetic relationship could not be resolved. Special attention was given to the differentiation between D. americana texana, D. americana americana and D. novamexicana. Significant differences between these three groups were detected by F(ST) analyses. Similarly, a model-based clustering method for multilocus genotype data also provided strong support for the presence of three differentiated groups. This genome-wide differentiation between D. americana texana and D. americana americana contrasts with previous analyses based on DNA sequence data.     

Chromosomal rearrangement In(2)TY and linkage maps of the second chromosome of Drosophila virilis

A chromosomal rearrangement In(2)TY, found in a natural population of Drosophila virilis, was examined cytologically, and its genetical characterization was carried out by the use of some marker isozymes and visible marker loci. From the interspecific hybridization between D. virilis and D. americana or D. texana, we deduced that the origin of In(2)TY was from a member of a closely related species of virilis.     

The relationships among the species of the Drosophila virilis group inferred from the gene Ras1 sequences

Comparative analysis of a group of closely related Drosophila species (D. virilis, D. lummei, D. novamexicana, D. americana texana, D. flavomontana, D. montana, D. borealis, D. lacicola, D. littoralis, D. kanekoi, and D. ezoana) was conducted based on an incomplete sequence of gene Ras1. The pattern of the relationships among the species corresponded to that expected from analysis of morphological and cytogenetic characters. Statistical data favoring neutrality of the substitutions examined in the Ras1 gene are presented. This character of the gene Ras1 evolution confers more reliability to reconstruction of phylogenetic relationships among closely related species. The resultant tree for main phylads of the group is as follows: (D. virilis, D. lummei, D. montana, D. ezoana).     

A genealogical view of chromosomal evolution and species delimitation in the Drosophila virilis species subgroup

Chromosomal arrangement was a historically important character used for defining taxonomic boundaries. The Drosophila virilis species group exhibits a series of chromosomal rearrangements, and the resulting differences among karyotypes were primary characters originally used to define taxa within the group. However, some chromosomally divergent forms have not been sufficiently resolved in phylogenetic reconstructions of DNA sequences from several nuclear genes. Sequences of mitochondrial regions have the potential for finer-scale resolution of closely related taxa; therefore, sequences of two mitochondrial genes were used to examine phylogenetic relationships within the chromosomally variable virilis subgroup. Sequences were obtained from multiple strains of the Palearctic species, D. virilis and D. lummei, and the Nearctic species, D. novamexicana and two chromosomal forms of D. americana. Analyses support the recent emergence of the different chromosomal forms in North America. However, none of these chromosomally divergent forms exhibit reciprocal monophyly of their mtDNA sequences, which is the requirement for attaining genealogical species status.     

The DAIBAM MITE element is involved in the origin of one fixed and two polymorphic Drosophila virilis phylad inversions

Chromosomal inversions can originate from breakage and repair by non-homologous end-joining. Nevertheless, they can also originate from ectopic recombination between transposable elements located on the same chromosome inserted in opposite orientations. Here, we show that a MITE element (DAIBAM), previously involved in the origin of one Drosophila americana polymorphic inversion, is also involved in the origin of one fixed inversion between D. virilis and D. americana and another D. americana polymorphic inversion. Therefore, DAIBAM is responsible for at least 20% of the chromosomal rearrangements that are observed within and between species of the virilis phylad (D. virilis, D. lummei, D. novamexicana and D. americana), having thus played a significant role in the chromosomal evolution of this group of closely related species.     

The relationships among the species of the <Emphasis Type="Italic">Drosophila virilis</Emphasis> group inferred from the gene <Emphasis Type="Italic">Ras1</Emphasis> sequences

Comparative analysis of a group of closely related Drosophila species (D. virilis, D. lummei, D. novamexicana, D. americana texana, D. flavomontana, D. montana, D. borealis, D. lacicola, D. littoralis, D. kanekoi, and D. ezoana) was conducted based on an incomplete sequence of gene Ras1. The pattern of the relationships among the species corresponded to that expected from analysis of morphological and cytogenetic characters. Statistical data favoring neutrality of the substitutions examined in the Ras1 gene are presented. This character of the gene Ras1 evolution confers more reliability to reconstruction of phylogenetic relationships among closely related species. The resultant tree for main phylads of the group is as follows: D. virilis (D. lummei, D. montana, D. ezoana).     

Species-specific differences in tissue-specific expression of alcohol dehydrogenase are under the control of complexcis-acting loci: Evidence fromDrosophila hybrids

Differences in the expression of alcohol dehydrogenase in the hindgut and testis of adult Drosophila virilis, D. texana, D. novamexicana and D. borealis flies were observed. These heritable differences do not arise due to chromosomal rearrangements, since the polytene chromosome banding patterns did not reveal any such gross chromosomal rearrangements near the Adh locus in any of the tested species. Analysis of the interspecific hybrids revealed that these differences are controlled by complex cis-acting genetic loci. Further, the cis-acting locus controlling the expression of ADH in testis was found to be separable by crossing-over.     

The Genetic Basis of a Species-Specific Character in the Drosophila Virilis Species Group

The genetic basis of the species-specific dorsal abdominal stripe of Drosophila novamexicana was examined. The dorsal stripe is present in D. novamexicana and absent in all other members of the Drosophila virilis species group. Interspecific crosses between D. novamexicana and genetically marked D. virilis revealed that all four of the autosomes (except the tiny dot chromosome, which was not marked) and the sex chromosomes (the X and Y chromosome effects could not be disentangled) showed a significant effect on the width of the dorsal stripe. All the autosomes act approximately additively; only minor interactions were detected among them. No significant maternal effects were found. This means that a minimum of five loci are involved in the character difference between the two species, and this is the maximum number that this technique could discern. These results suggest that, based on the number of factors involved in the character difference, the inheritance of this character should be considered polygenic, but because chromosome 2 (the largest chromosome in the species) contributed over half of the variance toward the character difference, it is best to consider the inheritance oligogenic based on effect. The implications of these findings are discussed in light of the importance of macromutation in speciation and the sex chromosome theory of speciation.     

Chromosomal and allelic variation in Drosophila americana: selective maintenance of a chromosomal cline.

Geographically structured genetic variation, as represented by clines and hybrid zones, offers unique opportunities to study adaptation and speciation in natural populations. A hybrid zone has been reported between Drosophila americana americana and Drosophila americana texana, two taxa that are distinguished solely by the arrangement of their X and 4th chromosomes. In this study, samples of D. americana were collected along a latitudinal transect across the inferred hybrid zone, and the frequency of the alternative chromosomal arrangements is reported. These data illustrate that the alternative chromosomal arrangements are distributed along a shallow cline over a broad geographic region, and that the frequency of the arrangements is tightly correlated with latitude. Allelic variants at 13 RFLP loci in three genes on chromosome 4 exhibit no evidence of association with the cline. Presence of a cline for the chromosomal arrangements, as well as a general absence of geographic structure for variation at these genes, is interpreted as evidence that natural selection is responsible for the maintenance of this chromosomal cline. Furthermore, these results demonstrate that taxonomic subdivision of D. americana is unwarranted, because it exists as a cohesive species that is segregating a chromosomal fusion.     

Puff expression in relation to genotype]

The studies of genotype influence on puff size in salivary gland chromosomes of Drosophila virilis (stocks 9, 101, 142, 151) and D. texana (the stock 123) reveal significant differences between the species concerning the structure of puff in the 3-C-6 region at the stage of puparium formation. In reciprocal F1 hybrids the size of the puff was intermediate in comparison with parental forms having a slight maternal effect. The differences in puff size in the 5th chromosome between interspecific hybrids and the special stock of D. virilis carrying a region of D. texana 5th chromosome in heterozygous condition (inserted into D. virilis 5the chromosome by double crossing-over) were observed. The transfer of the region of the third chromosome to near centrimetric heterochromatic of the 5th chromosome by translocation resulted in the increase in the 3-B-2 puff size. However, the transposition of the 3-B1 region in the proximal direction with respect to chromocenter did not affect the puff size.     

Patterns of molecular variation and evolution in Drosophila americana and its relatives

We present the results of a survey of DNA sequence variability at X-linked and autosomal loci in Drosophila americana and of patterns of DNA sequence evolution among D. americana and four other related species in the virilis group of Drosophila. D. americana shows a typical level of silent polymorphism for a Drosophila species, but has an unusually low ratio of nonsynonymous to silent variation. Both D. virilis and D. americana also show a low ratio of nonsynonymous to synonymous substitutions along their respective lineages since the split from their common ancestor. The proportion of amino acid substitutions between D. americana and its relatives that are caused by positive selection, as estimated by extensions of the McDonald-Kreitman test, appears to be unusually high. We cannot, however, exclude the possibility that this reflects a recent increase in the intensity of selection on nonsynonymous mutations in D. americana and D. virilis. We also find that base composition at neutral sites appears to be in overall equilibrium among these species, but there is evidence for departure from equilibrium for codon usage in some lineages.     

Lack of Degeneration of Loci on the Neo-Y Chromosome of Drosophila Americana Americana

The extent of genetic degeneration of the neo-Y chromosome of Drosophila americana americana has been investigated. Three loci, coding for the enzymes enolase, phosphoglycerate kinase and alcohol dehydrogenase, have been localized to chromosome 4 of D. a. americana, which forms the neo-Y and neo-X chromosomes. Crosses between D. a. americana and D. virilis or D. montana showed that the loci coding for these enzymes carry active alleles on the neo-Y chromosome in all wild-derived strains of americana that were tested. Intercrosses between a genetically marked stock of virilis and strains of americana were carried out, creating F(3) males that were homozygous for sections of the neo-Y chromosome. The sex ratios in the F(3) generation of the intercrosses showed that no lethal alleles have accumulated on any of the neo-Y chromosomes tested. There was evidence for more minor reductions in fitness, but this seems to be mainly caused by deleterious alleles that are specific to each strain. A similar picture was provided by examination of the segregation ratios of two marker genes among the F(3) progeny. Overall, the data suggest that the neo-Y chromosome has undergone very little degeneration, certainly not to the extent of having lost the functions of vital genes. This is consistent with the recent origin of the neo-Y and neo-X chromosomes, and the slow rates at which the forces that cause Y chromosome degeneration are likely to work.     

Heterochromatin in mitotic chromosomes of theVirilis species group ofDrosophila

The amount of heterochromatin in the genome of ten members of thevirilis species group was determined as the length of C-band chromosome material relative to the total karyotype length. Thevirilis phylad (Drosophila virilis, D. novamexicana, D. americana americana, andD. americana texana) has significantly greater amounts of heterochromatin in the genome than do members of the montana phylad (D. montana, D. lacicola, D. flavomontana, D. borealis, D. ezoana, D. littoralis). Thus, the significant karyotypic change accompanying diversification of these species has involved reduction in their total constitutive heterochromatin. These changes have apparently involved reductions in the amount of centromeric heterochromatin in the autosomes.     

Reduced sequence variability on the Neo-Y chromosome of Drosophila americana americana.

Sex chromosomes are generally morphologically and functionally distinct, but the evolutionary forces that cause this differentiation are poorly understood. Drosophila americana americana was used in this study to examine one aspect of sex chromosome evolution, the degeneration of nonrecombining Y chromosomes. The primary X chromosome of D. a. americana is fused with a chromosomal element that was ancestrally an autosome, causing this homologous chromosomal pair to segregate with the sex chromosomes. Sequence variation at the Alcohol Dehydrogenase (Adh) gene was used to determine the pattern of nucleotide variation on the neo-sex chromosomes in natural populations. Sequences of Adh were obtained for neo-X and neo-Y chromosomes of D. a. americana, and for Adh of D. a. texana, in which it is autosomal. No significant sequence differentiation is present between the neo-X and neo-Y chromosomes of D. a. americana or the autosomes of D. a. texana. There is a significantly lower level of sequence diversity on the neo-Y chromosome relative to the neo-X in D. a. americana. This reduction in variability on the neo-Y does not appear to have resulted from a selective sweep. Coalescent simulations of the evolutionary transition of an autosome into a Y chromosome indicate there may be a low level of recombination between the neo-X and neo-Y alleles of Adh and that the effective population size of this chromosome may have been reduced below the expected value of 25% of the autosomal effective size, possibly because of the effects of background selection or sexual selection.     

Sequence evolution of theGpdh gene in theDrosophila virilis species group

The nucleotide sequence of theGpdh gene from six taxa,D. virilis, D. lummei, D. novamexicana, D. a. americana, D. a. texana andD. ezoana, belonging to thevirilis species group was determined to examine details of evolutionary change in the structure of theGpdh gene. TheGpdh gene is comprised of one 5 non-translated region, eight exons, seven introns and three 3 non-translated regions. Exon/intron organization was identical in all the species examined, but different from that of mammals. Interspecific nucleotide divergence in the entireGpdh gene followed the common pattern: it was low in the exon, high in the intron and intermediate in the non-translated regions. The degree of nucleotide divergence differed within these regions, suggesting that selection exerts constraints differentially on nucleotide change of theGpdh gene. A phylogenetic tree of thevirilis phylad constructed from nucleotide variation of total sequence was consistent with those obtained from other data.Nucleotide sequences for theGpdh gene ofD. lummei, D. novamexicana, D. a. americana, D. a. texana andD. ezoana have been submitted to GenBank with accession numbers D50087, D50088, D50089, D50090 and D50091.     

Conjugation of polytene chromosomes in intraspecies hybrids of the virilis group of Drosophila. II. Hybridization of complementary RNA with polytene chromosomes in specimens]

A cytological hybridization of H-3-complementary RNA synthetized from DNA a template of D. virilis with the polytene chromosomes of D. virilis and the hybrids between D. virilis and D. texana, was carried out in situ. The uridine label of RNA was shown to be located mainly over the disc of the polytene chromosomes, the silver grains in interspecies hybrids being located over both homogous chromosomes including the unpaired gions.     

DNA sequence divergence in the Drosophila virilis group

DNA sequence divergence was analyzed in some sibling species of the Drosophila virilis group. Clones comprising about 0.1% of the genome DNA were selected at random from a D. virilis library for a comparative study on DNA from D. lummei, D. novamexicana, D. borealis, and D. lacicola. Blot hybridization experiments indicated that about 70% of DNA from D. lummei and D. novamexicana and less than 50% of DNA from D. borealis and D. lacicola share sequences that are homologous to DNA in D. virilis. This finding is in excellent agreement with the genealogical tree based on cytological studies (Throckmorton 1982). - Four plasmids with inserts which are present in one or a few copies per genome were hybridized in situ to polytene chromosomes. These experiments demonstrate that (1) homologous "unique" DNA sequences are localized exclusively in homologous bands and (2) homologous bands that appear to be identical in different species may contain different DNA sequences.     

Discordant Rates of Chromosome Evolution in the Drosophila Virilis Species Group

In Drosophila, the availability of polytene chromosome maps and of sets of probes covering most regions of the chromosomes allows a direct comparison of the organization of the genome in different species. In this work, we report the localization, in Drosophila virilis, D. montana, and D. novamexicana, of >100 bacteriophage P1 clones containing ~65 kilobase inserts of genomic DNA from D. virilis. Each clone hybridizes with a single euchromatic site in either chromosome 1 or chromosome 3 in D. virilis. From these data, it is possible to estimate the minimum number of inversions required to transform the map positions of the probes in one species into the map positions of the same probes in a related species. The data indicate that, in the D. virilis species group, the X chromosome has up to four times the number of inversions as are observed in chromosome 3. The first photographic polytene chromosome maps for D. montana and D. novamexicana are also presented.