Hybrid Genome Evolution by Transposition: An Update.

Contrary to the view that hybrids are lineages devoid of evolutionary value, a number of case studies that have been lately reported show how hybrids are at the origin of many species. Some well-documented cases demonstrate that bursts of transposition often follow hybridization, generating new genetic variability. Studies in hybrid transposition strongly suggest that epigenetic changes and divergence in piRNA pathways drive deregulation in TE landscapes. Here, I have focused on mechanisms acting in Drosophila hybrids between two cactophilic species. The results reported here show that while hybrid instability by transposition is a genome-wide event, deregulation by TE overexpression in hybrid ovaries is not a general rule. When piRNA pools of ovaries are studied, results show that TEs with parental differences higher than 2-fold in their piRNA amounts are not more commonly deregulated in hybrids than TEs with similar levels, partially discrediting the generality of the maternal cytotype hypothesis. Some promising results on the piRNA pathway global failure hypothesis, which states that accumulated divergence of piRNA effector proteins is responsible for hybrid TE deregulation, have also been obtained. Altogether, these results suggest that TE deregulation might be driven by several interacting mechanisms. A natural scenario is proposed in which genome instability by transposition leads to hybrid genome reorganization. Small hybrid populations, subjected to natural selection helped by genetic drift, evolve new adaptations adapted to novel environments. The final step is either introgression or even a new hybrid species.

Drosophila Females Undergo Genome Expansion after Interspecific Hybridization.

Genome size (or C-value) can present a wide range of values among eukaryotes. This variation has been attributed to differences in the amplification and deletion of different noncoding repetitive sequences, particularly transposable elements (TEs). TEs can be activated under different stress conditions such as interspecific hybridization events, as described for several species of animals and plants. These massive transposition episodes can lead to considerable genome expansions that could ultimately be involved in hybrid speciation processes. Here, we describe the effects of hybridization and introgression on genome size of Drosophila hybrids. We measured the genome size of two close Drosophila species, Drosophila buzzatii and Drosophila koepferae, their F1 offspring and the offspring from three generations of backcrossed hybrids; where mobilization of up to 28 different TEs was previously detected. We show that hybrid females indeed present a genome expansion, especially in the first backcross, which could likely be explained by transposition events. Hybrid males, which exhibit more variable C-values among individuals of the same generation, do not present an increased genome size. Thus, we demonstrate that the impact of hybridization on genome size can be detected through flow cytometry and is sex-dependent.

A genome-wide survey of genetic instability by transposition in Drosophila hybrids.

Hybridization between species is a genomic instability factor involved in increasing mutation rate and new chromosomal rearrangements. Evidence of a relationship between interspecific hybridization and transposable element mobilization has been reported in different organisms, but most studies are usually performed with particular TEs and do not discuss the real effect of hybridization on the whole genome. We have therefore studied whole genome instability of Drosophila interspecific hybrids, looking for the presence of new AFLP markers in hybrids. A high percentage (27-90%) of the instability markers detected corresponds to TEs belonging to classes I and II. Moreover, three transposable elements (Osvaldo, Helena and Galileo) representative of different families, showed an overall increase of transposition rate in hybrids compared to parental species. This research confirms the hypothesis that hybridization induces genomic instability by transposition bursts and suggests that genomic stress by transposition could contribute to a relaxation of mechanisms controlling TEs in the Drosophila genome.

Genome-wide dissection of hybrid sterility in Drosophila confirms a polygenic threshold architecture.

To date, different studies about the genetic basis of hybrid male sterility (HMS), a postzygotic reproductive barrier thoroughly investigated using Drosophila species, have demonstrated that no single major gene can produce hybrid sterility without the cooperation of several genetic factors. Early work using hybrids between Drosophila koepferae (Dk) and Drosophila buzzatii (Db) was consistent with the idea that HMS requires the cooperation of several genetic factors, supporting a polygenic threshold (PT) model. Here we present a genome-wide mapping strategy to test the PT model, analyzing serially backcrossed fertile and sterile males in which the Dk genome was introgressed into the Db background. We identified 32 Dk-specific markers significantly associated with hybrid sterility. Our results demonstrate 1) a strong correlation between the number of segregated sterility markers and males' degree of sterility, 2) the exchangeability among markers, 3) their tendency to cluster into low-recombining chromosomal regions, and 4) the requirement for a minimum number (threshold) of markers to elicit sterility. Although our findings do not contradict a role for occasional major hybrid-sterility genes, they conform more to the view that HMS primarily evolves by the cumulative action of many interacting genes of minor effect in a complex PT architecture.

Adaptation of the AFLP technique as a new tool to detect genetic instability and transposition in interspecific hybrids.

An adapted amplified fragment length polymorphism (AFLP) protocol is presented for detection of hybrid instability in the genome of interspecific hybrids between Drosophila buzzatii and D. koepferae species. Analyses of 15 AFLP instability markers (new bands detected in hybrids) show that up to 81% are the result of transposable element (TE) activity. Twenty TEs associated with AFLP instability markers have been detected by this method in backcross hybrids and segmental hybrids, demonstrating its validity in detecting transposition events occurring during the hybridization process. New insertions of Helena TE have been observed in the hybrid genome after hybridization of the TGTCG22 instability marker by FISH. The AFLP marker technique proved to be an efficient method that improves upon traditional and bioinformatic tools previously used to detect TE mobilization. This newly adapted AFLP protocol may also be applied to a large number of organisms outside the Drosophila genus, making it of interest to evolutionary and population genetic researchers working with species where the knowledge of the genome is scarce.

Osvaldo and Isis retrotransposons as markers of the Drosophila buzzatii colonisation in Australia.

BACKGROUND: Transposable elements (TEs) constitute an important source of genetic variability owing to their jumping and regulatory properties, and are considered to drive species evolution. Several factors that are able to induce TE transposition in genomes have been documented (for example environmental stress and inter- and intra-specific crosses) but in many instances the reasons for TE mobilisation have yet to be elucidated. Colonising populations constitute an ideal model for studying TE behaviour and distribution as they are exposed to different environmental and new demographic conditions. In this study, the distribution of two TEs, Osvaldo and Isis, was examined in two colonising populations of D. buzzatii from Australia. Comparing Osvaldo copy numbers between Australian and Old World (reported in previous studies) colonisations provides a valuable tool for elucidating the colonisation process and the effect of new conditions encountered by colonisers on TEs. RESULTS: The chromosomal distributions of Osvaldo and Isis retrotransposons in two colonising populations of D. buzzatii from Australia revealed sites of high insertion frequency (>10%) and low frequency sites. Comparisons between Osvaldo insertion profiles in colonising populations from the Old World and Australia demonstrate a tendency towards a higher number of highly occupied sites with higher insertion frequency in the Old World than in Australian populations. Tests concerning selection against deleterious TE insertions indicate that Isis is more controlled by purifying selection than Osvaldo. The distribution of both elements on chromosomal arms follows a Poisson distribution and there are non-significant positive correlations between highly occupied sites and chromosomal inversions. CONCLUSIONS: The occupancy profile of Osvaldo and Isis retrotransposons is characterised by the existence of high and low insertion frequency sites in the populations. These results demonstrate that Australian D. buzzatii populations were subjected to a founder effect during the colonisation process. Moreover, there are more sites with high insertion frequency in the Old World colonisation than in the Australian colonisation, indicating a probable stronger bottleneck effect in Australia. The results suggest that selection does not seem to play a major role, compared to demography, in the distribution of transposable elements in the Australian populations.

Distribution of the transposable elements bilbo and gypsy in original and colonizing populations of Drosophila subobscura.

BACKGROUND: Transposable elements (TEs) constitute a substantial amount of all eukaryotic genomes. They induce an important proportion of deleterious mutations by insertion into genes or gene regulatory regions. However, their mutational capabilities are not always adverse but can contribute to the genetic diversity and evolution of organisms. Knowledge of their distribution and activity in the genomes of populations under different environmental and demographic regimes, is important to understand their role in species evolution. In this work we study the chromosomal distribution of two TEs, gypsy and bilbo, in original and colonizing populations of Drosophila subobscura to reveal the putative effect of colonization on their insertion profile. RESULTS: Chromosomal frequency distribution of two TEs in one original and three colonizing populations of D. subobscura, is different. Whereas the original population shows a low insertion frequency in most TE sites, colonizing populations have a mixture of high (frequency > or = 10%) and low insertion sites for both TEs. Most highly occupied sites are coincident among colonizing populations and some of them are correlated to chromosomal arrangements. Comparisons of TE copy number between the X chromosome and autosomes show that gypsy occupancy seems to be controlled by negative selection, but bilbo one does not. CONCLUSION: These results are in accordance that TEs in Drosophila subobscura colonizing populations are submitted to a founder effect followed by genetic drift as a consequence of colonization. This would explain the high insertion frequencies of bilbo and gypsy in coincident sites of colonizing populations. High occupancy sites would represent insertion events prior to colonization. Sites of low frequency would be insertions that occurred after colonization and/or copies from the original population whose frequency is decreasing in colonizing populations. This work is a pioneer attempt to explain the chromosomal distribution of TEs in a colonizing species with high inversion polymorphism to reveal the putative effect of arrangements in TE insertion profiles. In general no associations between arrangements and TE have been found, except in a few cases where the association is very strong. Alternatively, founder drift effects, seem to play a leading role in TE genome distribution in colonizing populations.

The evolutionary history of Drosophila buzzatii. XXXVI. Molecular structural analysis of Osvaldo retrotransposon insertions in colonizing populations unveils drift effects in founder events.

Previous work on transposable element distribution in colonizing populations of Drosophila buzzatii revealed a high frequency of occupancy in several chromosomal sites. Two explanatory hypotheses were advanced: the founder hypothesis, by which founder genetic drift was responsible, and the unstable hypothesis that assigns this unusual distribution to bursts of transposition toward some chromosomal sites. Here, we study the molecular structure of three euchromatic Osvaldo clones isolated from sites occupied at high (A4 and B9) and low frequency (B4) in colonizing populations, to test these hypotheses. Large insertions, duplications, and indels in the Osvaldo coding region and LTR were detected in the A4 clone and a truncated Osvaldo with many substitutions was found in the B9 clone. These altered sequences indicate that the two copies of this retroelement are precolonization insertions. Interestingly, the LTR of the A4 clone and the reverse transcriptase region of B9 show identical sequences in all colonizing populations indicating, most probably, that they are identical by descent. Moreover, Osvaldo is inserted at the same nucleotide site in all colonizing populations. On the other hand an almost identical LTR sequence, except by 1 base deletion, was found in the B4 clone compared to the canonical active Osvaldo element. These results suggest that Osvaldo copies in highly occupied sites are, most probably, identical by descent and strongly favor the founder hypothesis. On the other hand, low-insertion-frequency sites could represent recent transposition events. This work emphasizes the importance of molecular population studies to disentangle the effects of genetic drift and transposition in colonization.

Phylogeny and molecular evolution of the Drosophila hydei subgroup (Drosophila repleta group) inferred from the Xanthine dehydrogenase gene.

The hydei subgroup (Drosophila repleta group) consists of seven species divided into two complexes: bifurca and hydei, whose phylogenetic relationships are not well understood. To evaluate the molecular phylogeny of this subgroup, we analyzed 2085 bp of coding sequence of the Xanthine dehydrogenase gene in six available species of the hydei subgroup, with Drosophila buzzatii and Drosophila mulleri as an outgroup. For phylogenetic reconstruction we adopted a maximum-likelihood framework, based on the adjustment of descriptive models of nucleotide substitution to real data. We employed distance-based and weighted parsimony methods to construct candidate phylogenies. In all cases, we obtained only one completely resolved tree with strong statistical support for each node, that shows a phylogeny that is partially discordant with the proposed systematics of the subgroup. This tree suggests that the two species complexes are paraphyletic, as opposed to classic phylogenies using morphologic and cytologic traits. This discordance is discussed in relation to its implication for the evolutionary history of the hydei subgroup.

Temperature-related genetic changes in laboratory populations of Drosophila subobscura: evidence against simple climatic-based explanations for latitudinal clines.

Parallel latitudinal clines to the long-standing ones in the original Palearctic populations have independently evolved at different rates for chromosomal polymorphism and body size in South and North American populations of Drosophila subobscura since colonization around 25 years ago. This strongly suggests that (micro) evolutionary changes are largely predictable, but the underlying mechanisms are unknown. The putative role of temperature per se was investigated by using three sets of populations at each of three temperatures (13 degrees , 18 degrees , and 22 degrees C) spanning much of the tolerable range for this species. We found a lower chromosomal diversity at the warmest temperature; a quick and consistent shift in gene arrangement frequencies in response to temperature; an evolutionary decrease in wing size, mediated by both cell area and cell number, at 18 degrees C; no relationship between wing size and those inversions involved in latitudinal clines; and a shortening of the basal length of longitudinal vein IV relative to its total length with increasing standard dose. The trends for chromosomal polymorphism and body size were generally inconsistent from simple climatic-based explanations of worldwide latitudinal patterns. The findings are discussed in the light of available information on D. subobscura and results from earlier thermal selection experiments with various Drosophila species.

The evolutionary history of Drosophila buzzatii. XXXV. Inversion polymorphism and nucleotide variability in different regions of the second chromosome.

Inversions are portions of a chromosome where the gene order is reversed relative to a standard reference orientation. Because of reduced levels of recombination in heterokaryotypes, inversions have a potentially important effect on patterns of nucleotide variability in those genomic regions close to, or included in, the inverted fragments. Here we report sequence variation at three anonymous regions (STSs) located at different positions in relation to second-chromosome inversion breakpoints in 29 isochromosomal lines derived from an Argentinean population of Drosophila buzzatii. In agreement with previous findings in Drosophila, gene flux (crossing over and/or gene conversion) between arrangements seems to appreciably increase as we approach the middle sections of inversion 2j, and patterns of nucleotide variability within, as well as genetic differentiation between chromosome arrangements, are comparable to those observed at the molecular marker outside the inverted fragments. On the other hand, nucleotide diversity near the proximal breakpoint of inversion 2j is reduced when contrasted with that found at the other regions, particularly in the case of derived inverted chromosomes. Using the data from the breakpoint, we estimate that the inversion polymorphism is approximately 1.63 N generations old, where N is the effective population size. An excess of low-frequency segregating polymorphisms is detected; mostly in the ancestral 2st arrangement and probably indicating a population expansion that predates the coalescent time of inversion 2j. Heterogeneity in mutation rates between the markers linked to the inversions may be sufficient to explain the different levels of nucleotide diversity observed. When considered in the context of other studies on patterns of variation relative to physical distance to inversion breakpoints, our data appear to be consistent with the conclusion that inversions are unlikely to be "long-lived" balanced polymorphisms.

COMPETITION AND GENOTYPE-BY-ENVIRONMENT INTERACTION IN NATURAL BREEDING SUBSTRATES OF DROSOPHILA.

Although empirical studies frequently suggest that genotype-by-environment (G X E) interaction can maintain genetic variation, very few data are available to test for the specific conditions necessary for the existence of a protected polymorphism (i.e., the property of persistence of an allele even when initially rare). Drosophila species live in patchy environments and their local population structure may be characterized to some extent by Levene's migration pattern, namely by a single pool of individuals that presumably mate at random and breed on discrete and ephemeral resources. We present here a field experiment that links Drosophila ecology and population genetics, which used the alcohol dehydrogenase (Adh) and α-glycerophosphate dehydrogenase (αGpdh) polymorphic loci in D. melanogaster flies raised from Opuntia ficus-indica fruits (prickly pears). The results show that there is density-dependent mortality in those fruits with a relatively high number of larvae (i.e., selection is "soft") and suggest that there is differential viability for αGpdh genotypes. Additionally, a pattern of G X E interaction for fitness values, which is fully compatible with the theoretical conditions required for the existence of a protected polymorphism, was found after weighting the fitness estimates by the relative contribution that each fruit makes to the total adult population. The strong association between AdhS and αGpdhF alleles suggests that the occurrence of the common cosmopolitan inversion In(2L)t in the population might be responsible for the negative frequency-dependent selection predicted by Levene's model when genetic variation persists in heterogeneous environments.

MATING PATTERN AND FITNESS-COMPONENT ANALYSIS ASSOCIATED WITH INVERSION POLYMORPHISM IN A NATURAL POPULATION OF DROSOPHILA BUZZATII.

Direct studies of mating success or mating pattern associated with Mendelian factors rarely have been carried out in nature. From the samples taken for the standard analyses of selection components, it is not usually possible to obtain the mating table, and only directional selection for male mating success can be detected. Both processes, mating pattern and differential mating probability, together with other fitness components, have been investigated for the inversion polymorphism of a natural population of the cactophilic species Drosophila buzzatii. Two independent samples of adult flies were collected: nonmating or single individuals (base population) and mating pairs (mating population). All individuals were karyotyped for the second and fourth chromosomes. A sequence of models with increasing simplicity was fitted to the data to test null hypotheses of no selection and random union of gametes and karyotypes. The main results were (1) no deviations from random mating were found; (2) differential mating probability was nonsignificant in both sexes; (3) inversion and karyotypic frequencies did not differ between sexes; and (4) karyotypic frequencies did not depart from Hardy-Weinberg expectations. These results are discussed in light of complementary evidence showing the need for interpreting with caution no-effect hypotheses such as the ones tested here. The use of complementary selective tests in these studies is suggested.