Tracking the origin of an invasive species: Drosophila subobscura in Argentina.

Biological invasions are excellent opportunities to study the evolutionary forces leading to the adaptation of a species to a new habitat. Knowledge of the introduction history of colonizing species helps tracking colonizing routes and assists in defining management strategies for invasive species. The Palearctic species Drosophila subobscura is a good model organism for tracking colonizations since it was detected in Chile and western North America three decades ago and later on in the Atlantic coast of Argentina. To unravel the origin of the Argentinean colonizers two populations have been analysed with several genetic markers. Chromosomal arrangements and microsatellite alleles found in Argentina are almost similar to those observed in Chile and USA. The lethal allelism test demonstrates that the lethal gene associated with the O(5) inversions in Argentina is identical to that found in Chile and USA, strongly supporting the hypothesis that all the American colonizing populations originated from the same colonization event. A secondary bottleneck is detected in the Argentinean populations and the genetic markers suggest that these populations originated from the invasion of 80-150 founding individuals from Chile.

Molecular characterization and genomic distribution of Isis: a new retrotransposon of Drosophila buzzatii.

A new transposable element, Isis, is identified as a LTR retrotransposon in Drosophila buzzatii. DNA sequence analysis shows that Isis contains three long ORFs similar to gag, pol and env genes of retroviruses. The ORF1 exhibits sequence homology to matrix, capsid and nucleocapsid gag proteins and ORF2 encodes a putative protease (PR), a reverse transcriptase (RT), an Rnase H (RH) and an integrase (IN) region. The analysis of a putative env product, encoded by the env ORF3, shows a degenerated protein containing several stop codons. The molecular study of the putative proteins coded by this new element shows striking similarities to both Ulysses and Osvaldo elements, two LTR retrotransposons, present in D. virilis and D. buzzatii, respectively. Comparisons of the predicted Isis RT to several known retrotransposons show strong phylogenetic relationships to gypsy-like elements, particulary to Ulysses retrotransposon. Studies of Isis chromosomal distribution show a strong hybridization signal in centromeric and pericentromeric regions, and a scattered distribution along all chromosomal arms. The existence of insertional polymorphisms between different strains and high molecular weight bands by Southern blot suggests the existence of full-sized copies that have been active recently. The presence of euchromatic insertion sites coincident between Isis and Osvaldo could indicate preferential insertion sites of Osvaldo element into Isis sequence or vice versa. Moreover, the presence of Isis in different species of the buzzatii complex indicates the ancient origin of this element.

Hybrid genome evolution by transposition.

Species hybridization is reviewed focusing on its role as a source of evolutionary novelties. Contrary to the view that hybrids are lineages devoid of evolutionary value, a number of case studies are given that show how hybrids are responsible for reticulate evolution that may lead to the origin of new species. Hybrid evolution is mediated by extensive genome repatterning followed by rapid stabilization and fixation of highly adapted genotypes. Some well-documented cases demonstrate that bursts of transposition follow hybridization and may contribute to the genetic instability observed after hybridization. The mechanism that triggers transposition in hybrids is largely unknown, but coupling of hybrid transposition and demethylation has been observed in mammals and plants. A natural scenario is proposed in which marginal small hybrid populations undergo transposition mediated genome reorganizations accompanied by exogenous and endogenous selection that, in concert with drift, lead to rapid fixation of high fitness hybrid genotypes. These genotypes may represent parental introgressed species or be entirely new species.

Swift laboratory thermal evolution of wing shape (but not size) in Drosophila subobscura and its relationship with chromosomal inversion polymorphism.

Latitudinal clinal variation in wing size and shape has evolved in North American populations of Drosophila subobscura within about 20 years since colonization. While the size cline is consistent to that found in original European populations (and globally in other Drosophila species), different parts of the wing have evolved on the two continents. This clearly suggests that 'chance and necessity' are simultaneously playing their roles in the process of adaptation. We report here rapid and consistent thermal evolution of wing shape (but not size) that apparently is at odds with that suggestion. Three replicated populations of D. subobscura derived from an outbred stock at Puerto Montt (Chile) were kept at each of three temperatures (13, 18 and 22 degrees C) for 1 year and have diverged for 27 generations at most. We used the methods of geometric morphometrics to study wing shape variation in both females and males from the thermal stocks, and rates of genetic divergence for wing shape were found to be as fast or even faster than those previously estimated for wing size on a continental scale. These shape changes did not follow a neat linear trend with temperature, and are associated with localized shifts of particular landmarks with some differences between sexes. Wing shape variables were found to differ in response to male genetic constitution for polymorphic chromosomal inversions, which strongly suggests that changes in gene arrangement frequencies as a response to temperature underlie the correlated changes in wing shape because of gene-inversion linkage disequilibria. In fact, we also suggest that the shape cline in North America likely predated the size cline and is consistent with the quite different evolutionary rates between inversion and size clines. These findings cast strong doubts on the supposed 'unpredictability' of the geographical cline for wing traits in D. subobscura North American colonizing populations.

Chromosomal distribution of the transposable elements Osvaldo and blanco in original and colonizer populations of Drosophila buzzatii.

Chromosomal distribution of transposable elements (TEs) Osvaldo and blanco in D. buzzatii was studied in three original natural populations from Argentina (Berna, Puerto Tirol and La Nostalgia) and a colonizer population from the Iberian Peninsula (Carboneras). The Spanish population showed significant differences for Osvaldo and blanco copy numbers when we compared the X chromosome and the autosomes; but it is mainly the accumulation of copies in chromosome 2, where most sites with high insertion frequency were located, that causes the discrepancy with the negative selection model. We found no significant differences in TE frequency between chromosomal regions with different exchange rates, and no evident accumulation of TE was detected within chromosomal inversions where recombination rate is reduced. The Carboneras population shows euchromatic sites of Osvaldo and blanco with high occupancy and others with low copy number. On the contrary, Argentinian populations show only a generalized low occupancy per insertion site. Moreover, the mean copy number of both elements is higher in Spain than in Argentina. All these results suggest an important role of the colonization process in the distribution of TEs. The increase in the copy number of the TEs analysed and their elevated frequency in some chromosomal sites in Carboneras is, most probably, a sequel of the founder event and drift that took place at the time of the colonization of the Old World by D. buzzatii from the New World some 300 years ago.

Toward a physical map of Drosophila buzzatii. Use of randomly amplified polymorphic dna polymorphisms and sequence-tagged site landmarks.

We present a physical map based on RAPD polymorphic fragments and sequence-tagged sites (STSs) for the repleta group species Drosophila buzzatii. One hundred forty-four RAPD markers have been used as probes for in situ hybridization to the polytene chromosomes, and positive results allowing the precise localization of 108 RAPDs were obtained. Of these, 73 behave as effectively unique markers for physical map construction, and in 9 additional cases the probes gave two hybridization signals, each on a different chromosome. Most markers (68%) are located on chromosomes 2 and 4, which partially agree with previous estimates on the distribution of genetic variation over chromosomes. One RAPD maps close to the proximal breakpoint of inversion 2z(3) but is not included within the inverted fragment. However, it was possible to conclude from this RAPD that the distal breakpoint of 2z(3) had previously been wrongly assigned. A total of 39 cytologically mapped RAPDs were converted to STSs and yielded an aggregate sequence of 28,431 bp. Thirty-six RAPDs (25%) did not produce any detectable hybridization signal, and we obtained the DNA sequence from three of them. Further prospects toward obtaining a more developed genetic map than the one currently available for D. buzzatii are discussed.

Molecular evolution and phylogeny of the buzzatii complex (Drosophila repleta group): a maximum-likelihood approach.

The buzzatii complex of the mulleri subgroup (Drosophila repleta group) consists of three clusters of species whose evolutionary relationships are poorly known. We analyzed 2,085 coding nucleotides from the xanthine dehydrogenase (XDH:) gene in the 10 available species of the complex and Drosophila mulleri and Drosophila hydei. We adopted a statistical model-fitting approach within the maximum-likelihood (ML) framework of phylogenetic inference. We first modeled the process of nucleotide substitution using a tree topology which was reasonably accurate. Then we used the most satisfactory description so attained to reconstruct the evolutionary relationships in the buzzatii complex. We found that a minimally realistic description of the substitution process of XDH: should allow six substitution types and different substitution rates for codon positions. Using this description we obtained a strongly supported, fully resolved tree which is congruent with the already-known (yet few) relationships. We also analyzed published data from three mitochondrial cytochrome oxidases (CO I, II, and III). In our analyses, these relatively short DNA sequences failed to discriminate statistically among alternative phylogenies. When the data of these three gene regions are combined with the XDH: sequences, the phylogenetic signal emerging from XDH: becomes reinforced. All four of the gene regions evolve faster in the buzzatii and martensis clusters than in the stalkeri cluster, paralleling the amount of chromosomal evolution.

The retrotransposon Osvaldo from Drosophila buzzatii displays all structural features of a functional retrovirus.

The Osvaldo retrotransposon has shown a high transposition rate in some strains of Drosophila buzzatii and in hybrids between D. buzzatii and its sibling D. koepferae. In order to understand the molecular basis of this phenomenon, we developed a procedure to clone a recently transposed copy with the aim of characterizing an active, full- length Osvaldo element. The complete nucleotide sequence of Osvaldo, obtained from a recent insertion site, was determined. Osvaldo is 9,045 bp long and is composed of a central coding region flanked by identical long terminal repeats (LTRs) of 1,196 bp each. Sequences homologous to the polypurine tract and tRNA-primer-binding site of retroviruses are located adjacent to the 3' and 5' LTRs, respectively. The internal region of Osvaldo contains three long open reading frames (ORFs 1, 2, and 3), comparable in size and location to gag, pol, and env retroviral genes. The conceptual translation of Osvaldo ORF1 exhibits sequence homology to HIV1 and SIV capsid (p24) and nucleocapsid (p7) mature proteins. ORF2 encodes the putative protease (PR), reverse transcriptase/ribonuclease H (RT/RH), integrase (IN), and a significant portion of the surface envelope (ENV) protein that is interrupted by a putative intron. A third ORF encodes the remaining part of the ENV protein. The predicted 62-kDa ENV protein shares several general features with membrane glycoproteins, including a potential signal peptide, a transmembrane domain near the C-terminus that could function as a membrane anchor, four consensus N-linked glycosylation motifs, and, finally, a potential protease cleavage site. The phylogenetic relationships of Osvaldo are explored, and they suggest that Osvaldo may constitute a new family of retroviruses in insects, distantly related to the previously described group of gypsy retroviruses.

Interspecific hybridization increases transposition rates of Osvaldo.

Several authors have postulated that genetic divergence between populations could result in genomic incompatibilities that would cause an increase in transposition in their hybrids, producing secondary effects such as sterility and therefore starting a speciation process. It has been demonstrated that transposition largely depends on intraspecific hybridization for P, hobo, and I elements in Drosophila melanogaster and for several elements, including long terminal repeat (LTR) and non-LTR retrotransposons, in D. virilis. However, in order to demonstrate the putative effect of transposable elements on speciation, high levels of transposition should also be induced in hybrids between species that could have been originated by this process and that are still able to interbreed. To test this hypothesis, we studied the transposition of the LTR retrotransposon Osvaldo in Drosophila buzzatii-Drosophila koepferae hybrids. We used a simple and robust experimental design, analyzing large samples of single-pair mate offspring, which allowed us to detect new insertions by in situ hybridization to polytene chromosomes. In order to compare transposition rates, we also used a stock recently obtained from the field and a highly inbred D. buzzatii strain. Our results show that the transposition rate of Osvaldo is 10(-3) transpositions per element per generation in all nonhybrid samples, very high when compared with those of other transposable elements. In hybrids, the transposition rate was always 10(-2), significantly higher than in nonhybrids. We show that inbreeding has no effect on transposition in the strains used, concluding that hybridization significantly increases the Osvaldo transposition rate.

Stable Drosophila buzzatii-Drosophila koepferae hybrids.

Previous experiments discovered high rates of chromosomal rearrangements in the progeny of males containing a telomeric segment of Drosophila koepferae in a D. buzzatii genetic background (segmental males). We have performed similar experiments, designed to test whether this chromosomal instability could be explained by a phenomenon similar to P-M hybrid dysgenesis or, alternatively, by a generalized telomeric effect. However, the results obtained have not allowed us to fully characterize this process, because we have not observed chromosomal rearrangements in the progeny of the putative unstable males. Our results suggest that chromosomal instability is independent of the introgressed fragment. A reasonable hypothesis to explain these results is that mutator factors are occasionally introduced by the hybridization. The effect of sampling, caused by the fact that only a small region is introgressed in a particular line, may explain why only some hybridizations lead to instability.

Evolutionary relationships among the members of an ancient class of non-LTR retrotransposons found in the nematode Caenorhabditis elegans.

We took advantage of the massive amount of sequence information generated by the Caenorhabditis elegans genome project to perform a comprehensive analysis of a group of over 100 related sequences that has allowed us to describe two new C. elegans non-LTR retrotransposons. We named them Sam and Frodo. We also determined that several highly divergent subfamilies of both elements exist in C. elegans. It is likely that several master copies have been active at the same time in C. elegans, although only a few copies of both Sam and Frodo have characteristics that are compatible with them being active today. We discuss whether it is more appropriate under these circumstances to define only 2 elements corresponding to the most divergent groups of sequences or up to 16, considering each subfamily a different element. The C. elegans elements are related to other previously described non-LTR retrotransposons (CR1, found in different vertebrates; SR1, from the trematode Schistosoma; Q and T1, from the mosquito Anopheles). All of these elements, according to the analysis of their reverse transcriptases, form a monophyletic cluster that we call the "T1/CR1 subgroup." Elements of this subgroup are thus ancient components of the genome of animal species. However, we discuss the possibility that these elements may occasionally be horizontally transmitted.

The evolutionary history of Drosophila buzzatii. XXXIV. The distribution of the retrotransposon Osvaldo in original and colonizing populations.

The frequency distribution of the retrotransposon Osvaldo in the haploid genome of Drosophila buzzatii has been studied in five natural populations from the Iberian Peninsula and six natural populations from Argentina. In Iberian populations, Osvaldo insertion sites do not follow a Poisson distribution, most probably due to eight euchromatic sites with high occupancy, found in all populations. The estimated alpha and beta parameters, which measure the relative importance of drift and negative selection in shaping frequency distributions, indicate that drift is the main force acting upon the distribution of Osvaldo in natural populations of D. buzzatii in the Iberian Peninsula. On the other hand, Osvaldo distribution in populations from Argentina is similar to the distribution of elements with low copy numbers, such as those described for Drosophila melanogaster and Drosophila simulans: there are no indications for deviation from a Poisson distribution, there is a low occupancy per insertion site, and genetic drift has no apparent effect on the frequency distribution. We propose that the unusual distribution found in the populations from the Iberian Peninsula is a consequence of the colonization process. Iberian Peninsula populations suffered a genomic redistribution of Osvaldo, most probably after a founder effect. Consequently, certain copies that arrived at high frequencies are showing a high occupancies today, and the mean copy number of Osvaldo is higher in Iberian Peninsula populations than in populations from Argentina. All other copies are the result of recent (after colonization) transposition events.

The evolutionary history of Drosophila buzzatii. XXXIII. Are Opuntia hosts a selective factor for the inversion polymorphism?

Previous work has shown fitness differences among chromosomal arrangements by means of selection component analysis in two Drosophila buzzatii natural populations, one of which is native to Argentina and the other a colonized population from Carboneras, Spain. Founder effects or niche shifts were proposed to explain the differences observed in the pattern of pleiotropic effects of inversions on fitness components. In this paper, we address the possible role of niche shifts by determining whether differential attraction to, oviposition on, or utilization of the rotting cladodes of two different Opuntia species (O. quimilo and O. ficus-indica) occurred among individuals carrying different second chromosome karyotypes in a natural Argentinian population. Through the analysis of more than 2500 individuals comprising five different life cycle stages associated with the necroses of these two cactus species, we found that the distributions of inversion frequencies in samples of adult flies, third instar larvae and emerging adults collected on both Opuntia species were not significantly different. Likewise, no evidence of differential oviposition was observed. These findings suggest that niche shifts cannot, solely, account for the changes observed in the Carboneras population. In addition, the selection component analysis did not reveal any significant relationship between chromosomal arrangements and the fitness components tested. These results suggest either that fitness differences might be too small to be detected or that the assumptions of the model concerning the mode of selection may not be tenable in the studied population.

Evolutionary conservation and molecular characteristics of repetitive sequences of Drosophila koepferae.

Thirteen middle repetitive DNA clones obtained from the genome of Drosophila koepferae have been tested for their evolutionary conservation in the other seven species of the buzzatii and martensis clusters (repleta group). All but two of these clones exhibit qualitatively similar patterns of hybridization in the eight species. The average interspecific hybridization signal is 85 per cent of that found intraspecifically, ranging from 73 to 93 per cent. Partial sequencing of six of these clones has shown sequences related to the retrotransposon Gypsy, first characterized in D. melanogaster, as well as to the Anopheles gambiae LINE elements T1Ag and Q. A fragment of a hitherto unknown, short inverted repeat transposable element has also been found. The evolutionary conservation of repetitive D. koepferae sequences seems to be related to the high proportion of simple DNA and inactive mobile elements in the genome of this species.

The evolutionary history of Drosophila buzzatii. XXX. Mitochondrial DNA polymorphism in original and colonizing populations.

Both original and colonizer populations of Drosophila buzzatii have been analyzed for mtDNA restriction polymorphisms. Most of the mtDNA nucleotide variation in original populations of NW Argentina can be explained by intrapopulation diversity and only a small fraction can be accounted for by between-population diversity. Similar results are obtained using either the estimated number of nucleotide substitutions per site or considering each restriction site as a locus. Colonizer populations of the Iberian Peninsula are monomorphic and show only the most common haplotype from the original populations. Under the infinite island model and assuming that populations are in equilibrium, fixation indices indicate enough gene flow to explain why the populations are not structured. Yet, the possibility exists that populations have not reached an equilibrium after a founder event at the end of the last Pleistocene glaciation. Tajima's test suggests that directional selection and/or a recent bottleneck could explain the present mtDNA differentiation. Considering the significant population structure found for the chromosomal and some allozyme polymorphisms, the among-population uniformity for mtDNA variability argues in favor of the chromosomal and some allozyme polymorphisms being adaptive.

Heritability and selection on body size in a natural population of Drosophila buzzatii.

An attempt was made to assess whether the phenotypic differences in body size (as measured by wing length) between wild-caught mating and single Drosophila buzzatii males could be attributed to genetic differences between the samples. Mating males were found to be larger and less variable than a random sample of the population. The progeny of the mating males (produced by crossing to a random female from a stock derived from the same population) were on average larger than those of the single males, but not significantly so (P = 0.063), and less phenotypically variable. This difference in variance between the samples suggests that there are indeed genetic differences between the paternal samples but tests for significant differences in the additive genetic component of variance proved inconclusive. For both samples it was found that while the ratio of additive genetic variation in the laboratory to phenotypic variation in the field yielded estimates of hs2(N) congruent to 10% the regression of offspring reared in the laboratory on parents from the wild was not significantly different from zero. In addition, it was found that the average development time of the progeny of the mating males is shorter than that of the random sample.

Characterization of Gandalf, a new inverted-repeat transposable element of Drosophila koepferae.

The cloning and characterization of Gandalf, a new DNA-transposing mobile element obtained from the Drosophila koepferae (repleta group) genome is described. A fragment of Gandalf was found in a middle repetitive clone that shows variable chromosomal localization. Restriction, Southern blot, PCR and sequencing analyses have shown that most Gandalf copies are about 1 kb long, are flanked by 12 bp inverted terminal repeats and contain subterminal repetitive regions on both sides of the element. As with other elements of the DNA-transposing type (known as the 'Ac family'), the Gandalf element generates 8 bp direct duplications at the insertion point. Coding region analysis has shown that the longer open reading frame found in Gandalf copies could encode part of a protein. However, whether or not the 1 kb copies of the element are actually the active transposons remains to be elucidated. Gandalf shows a very low copy number in D. buzzatii, a sibling species of D. koepferae. An attempt to induce interspecific hybrid dysgenesis in hybrids of these two species has been unsuccessful.

The evolutionary history of Drosophila buzzatii. XXXII. Linkage disequilibrium between allozymes and chromosome inversions in two colonizing populations.

Chromosome polymorphism in Drosophila buzzatii is under selection but the genes responsible for the effect of the inversions of fitness are unknown. On the other hand, there is evidence for selection on several allozyme loci but the presence of paracentric inversions on the second chromosome, where most of the polymorphic loci are located, complicates the interpretation. Studies of the associations between allozymes and inversions are thus necessary to help understand the effect of selection at both the chromosomal and allozymic level. Until now this kind of information has only been available in D. buzzatii for two loci, Est-1 and Est-2, in Australian populations. Here we describe the genetic constitution of two Old World populations, Carboneras and Colera. Emphasis has been placed on the analysis of the linkage disequilibria between the second chromosome arrangements and three allozyme loci, Est-2, Pept-2 and Aldox, located on this chromosome. In addition, the recombination frequencies between the loci, and between the loci and the inversion breakpoints, have been estimated and a genetic map of the three loci has been produced. The two populations differ in allele and arrangement frequencies, as well as in the pattern of one-locus disequilibria. Est-2 and Aldox are associated with the second chromosome arrangements in both populations. On the other hand, Pept-2 is associated with the inversions in Colera but not in Carboneras. The gametic associations among the three loci are discussed taking into account the position of these loci on the chromosome map and the lack of recombination in the heterokaryotypes.

High transposition rates of Osvaldo, a new Drosophila buzzatii retrotransposon.

Transposition of a new Drosophila retrotransposon was investigated. Total genomic Southern analysis and polytene in situ hybridizations in D. buzzatii strains and other related species using a 6 kb D. buzzatii clone (cDb314) showed a dispersed, repetitive DNA pattern, suggesting that this clone contains a transposable element (TE). We have sequenced the cDb314 clone and demonstrated that it contains all the conserved protein sequences and motifs typical of retrovirus-related sequences. Although cDb314 does not include the complete TE, the protein sequence alignment demonstrates that it includes a defective copy of a new long terminal repeat (LTR) retrotransposon, related to the gypsy family, which we have named Osvaldo. Using a D. buzzatii inbred line in which all insertion sites are known, we have measured Osvaldo transposition rates in hybrids between this D. buzzatii line and its sibling species D. koepferae. The results show that Osvaldo transposes in bursts at high rate, both in the D. buzzatii inbred line and in species hybrids.

The evolutionary history of Drosophila buzzatii. XXVII. Thorax length is positively correlated with longevity in a natural population from Argentina.

The correlation between body size and longevity was tested in an Argentinian natural population of Drosophila buzzatii. Mean thorax length of flies newly emerging from rotting cladodes of Opuntia vulgaris was significantly smaller than that of two samples of flies caught at baits. The present results which might be interpreted as directional selection for longevity favoring larger flies are in agreement with previous results achieved in a Spanish natural population of D. buzzatii. Flies emerging from different substrates showed significant differences in thorax length, suggesting that an important fraction of phenotypic variance can be attributed to environmental variability. However, laboratory and field work in different populations of D. buzzatii showed a significant genetic component for thorax length variation.