Sequence signatures of a recent chromosomal rearrangement in <Emphasis Type="Italic">Drosophila mojavensis</Emphasis>

The X-chromosome inversion, Xe, distinguishes Drosophila mojavensis and D. arizonae. Earlier work mapped the breakpoints of this inversion to large intervals and provided hypotheses for the locations of the breakpoints within 3000-bp intergenic regions on the D. mojavensis genome sequence assembly. Here, we sequenced these regions directly in the putatively ancestral D. arizonae X-chromosome. We find that the two inversion breakpoints are near an inverted gene duplication and a common repetitive element, respectively, and these features were likely present in the non-inverted ancestral chromosome on the D. mojavensis lineage. Contrary to an earlier hypothesis, the inverted gene duplication appears to predate the inversion. We find no sequence similarity between the breakpoint regions in the D. mojavensis ancestor, excluding an ectopic-exchange model of chromosome rearrangements. We also found no evidence that staggered single-strand breaks caused the inversion. We suggest these features may have contributed to the chromosomal breakages resulting in this inversion. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Comparative polytene chromosome maps of <Emphasis Type="Italic">D. montana</Emphasis> and <Emphasis Type="Italic">D. virilis</Emphasis>

Chromosomal inversion polymorphism was characterized in Finnish Drosophila montana populations. A total of 14 polymorphic inversions were observed in Finnish D. montana of which nine had not been described before. The number of polymorphic inversions in each chromosome was not significantly different from that expected, assuming equal chance of occurrence in the euchromatic genome. There was, however, no correlation between the number of polymorphic inversions and that of fixed inversions in each chromosome. Therefore, a simple neutral model does not explain the evolutionary dynamics of inversions. Furthermore, in contrast to results obtained by others, no significant correlation was found between the two transposable elements (TEs) Penelope and Ulysses and inversion breakpoints in D. montana. This result suggests that these TEs were not involved in the creation of the polymorphic inversions seen in D. montana. A comparative analysis of D. montana and Drosophila virilis polytene chromosomes 4 and 5 was performed with D. virilis bacteriophage P1 clones, thus completing the comparative studies of the two species.     

Differential Rates of Male Genital Evolution in Sibling Species of <Emphasis Type="Italic">Drosophila</Emphasis>

Genital morphology in animals with internal fertilization is considered to be among the fastest evolving traits. Sexual selection is often proposed as the main driver of genital diversification but the exact selection mechanisms involved are usually unclear. In addition, the mechanisms operating may differ even between pairs of sibling species. We investigated patterns of male genital variation within and between natural populations of the cactophilic fly Drosophila koepferae ranging its entire geographic distribution and compared them with those previously observed in its sibling species, D. buzzatii. Using both mtDNA and nDNA markers we found that genital shape variation in D. koepferae is more restricted than expected for neutral evolution, suggesting the predominance of stabilizing selection. We also detected dissimilar patterns of divergence between populations of D. koepferae that were allopatric and sympatric with D. buzzatii. The constrained evolution inferred for D. koepferae’s genitalia clearly contrasts with the rapid divergence and higher morphological disparity observed in the populations of D. buzzatii. Finally, different possible scenarios of male genital evolution in each species and within the radiation of D. buzzatii cluster are discussed.     

Experimental Evolution of Alkaloid Tolerance in Sibling <Emphasis Type="Italic">Drosophila</Emphasis> Species with Different Degrees of Specialization

Drosophila buzzatii and Drosophila koepferae are sibling species with marked ecological differences related to their patterns of host exploitation. D. buzzatii is a polyphagous species with a sub-cosmopolitan distribution, while D. koepferae is endemic to the mountain plateaus of the Andes, where it exploits alkaloidiferous columnar cacti as primary hosts. We use experimental evolution to study the phenotypic response of these cactophilic Drosophila when confronting directional selection to cactus chemical defenses for 20 generations. Flies adapted to cactus diets also experienced higher viability on alkaloid-enriched media, suggesting the selection of adaptive genetic variation for chemical-stress tolerance. The more generalist species D. buzzatii showed a rapid adaptive response to moderate levels of secondary metabolites, whereas the columnar cacti specialist D. koepferae tended to maximize fitness under harder conditions. The evolutionary dynamic of fitness-related traits suggested the implication of metabolic efficiency as a key mediator in the adaptive response to chemical stress. Although we found no evidence of adaptation costs accompanying specialization, our results suggest the involvement of compensatory evolution. Overall, our study proposes that differential adaptation to secondary metabolites may contribute to varying degrees of host specialization, favoring niche partitioning among these closely related species.     

Mating success depends on rearing substrate in cactophilic <Emphasis Type="Italic">Drosophila</Emphasis>

Drosophila buzzatii and D. koepferae coexist in the arid lands of southern South America and exploit different types of cactus as breeding hosts. The former prefers to lay eggs on the rotting pads of prickly pears (genus Opuntia) whereas D. koepferae exhibits greater acceptance for columnar cacti (e.g., Echinopsis terschekii). Here, we demonstrate that the rearing cacti affect male mating success, flies reared in each species’ preferred host exhibited enhanced mating success than those raised in secondary hosts. Opuntia sulphurea medium endows D. buzzatii males with greater mating ability while D. koepferae males perform better when flies develop in Echinopsis terschekii. These effects are not mediated through body size, even in D. buzzatii whose body size happens to be affected by the rearing cacti. This scenario, which is consistent with the evolution of host specialization and speciation through sensory drive, emphasizes the importance of habitat isolation in the coexistence of these cactophilic Drosophila.     

The Transposon Galileo Generates Natural Chromosomal Inversions in Drosophila by Ectopic Recombination

Background

Transposable elements (TEs) are responsible for the generation of chromosomal inversions in several groups of organisms. However, in Drosophila and other Dipterans, where inversions are abundant both as intraspecific polymorphisms and interspecific fixed differences, the evidence for a role of TEs is scarce. Previous work revealed that the transposon Galileo was involved in the generation of two polymorphic inversions of Drosophila buzzatii.

Methodology/Principal Findings

To assess the impact of TEs in Drosophila chromosomal evolution and shed light on the mechanism involved, we isolated and sequenced the two breakpoints of another widespread polymorphic inversion from D. buzzatii, 2z ³. In the non inverted chromosome, the 2z ³ distal breakpoint was located between genes CG2046 and CG10326 whereas the proximal breakpoint lies between two novel genes that we have named Dlh and Mdp. In the inverted chromosome, the analysis of the breakpoint sequences revealed relatively large insertions (2,870-bp and 4,786-bp long) including two copies of the transposon Galileo (subfamily Newton), one at each breakpoint, plus several other TEs. The two Galileo copies: (i) are inserted in opposite orientation; (ii) present exchanged target site duplications; and (iii) are both chimeric.

Conclusions/Significance

Our observations provide the best evidence gathered so far for the role of TEs in the generation of Drosophila inversions. In addition, they show unequivocally that ectopic recombination is the causative mechanism. The fact that the three polymorphic D. buzzatii inversions investigated so far were generated by the same transposon family is remarkable and is conceivably due to Galileo''s unusual structure and current (or recent) transpositional activity.     

Evolution of male genitalia: environmental and genetic factors affect genital morphology in two <Emphasis Type="Italic">Drosophila</Emphasis> sibling species and their hybrids

Background  

The rapid evolution of genital morphology is a fascinating feature that accompanies many speciation events. However, the underlying patterns and explanatory processes remain to be settled. In this work we investigate the patterns of intraspecific variation and interspecific divergence in male genitalic morphology (size and shape) in the cactophilic sibling species Drosophila buzzatii and D. koepferae. Genital morphology in interspecific hybrids was examined and compared to the corresponding parental lines.     

<Emphasis Type="Italic">Drosophila</Emphasis><Emphasis Type="Italic">P</Emphasis> transposons of the urochordata <Emphasis Type="Italic">Ciona intestinalis</Emphasis>

P transposons belong to the eukaryotic DNA transposons, which are transposed by a cut and paste mechanism using a P-element-coded transposase. They have been detected in Drosophila, and reside as single copies and stable homologous sequences in many vertebrate species. We present the P elements Pcin1, Pcin2 and Pcin3 from Ciona intestinalis, a species of the most primitive chordates, and compare them with those from Ciona savignyi. They showed typical DNA transposon structures, namely terminal inverted repeats and target site duplications. The coding region of Pcin1 consisted of 13 small exons that could be translated into a P-transposon-homologous protein. C. intestinalis and C. savignyi displayed nearly the same phenotype. However, their P elements were highly divergent and the assumed P transposase from C. intestinalis was more closely related to the transposase from Drosophila melanogaster than to the transposase of C. savignyi. The present study showed that P elements with typical features of transposable DNA elements may be found already at the base of the chordate lineage. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

A large inversion in the linear chromosome of <Emphasis Type="Italic">Streptomyces griseus</Emphasis> caused by replicative transposition of a new Tn<Emphasis Type="Italic">3</Emphasis> family transposon

We have comprehensively analyzed the linear chromosomes of Streptomyces griseus mutants constructed and kept in our laboratory. During this study, macrorestriction analysis of AseI and DraI fragments of mutant 402-2 suggested a large chromosomal inversion. The junctions of chromosomal inversion were cloned and sequenced and compared with the corresponding target sequences in the parent strain 2247. Consequently, a transposon-involved mechanism was revealed. Namely, a transposon originally located at the left target site was replicatively transposed to the right target site in an inverted direction, which generated a second copy and at the same time caused a 2.5-Mb chromosomal inversion. The involved transposon named TnSGR was grouped into a new subfamily of the resolvase-encoding Tn3 family transposons based on its gene organization. At the end, terminal diversity of S. griseus chromosomes is discussed by comparing the sequences of strains 2247 and IFO13350.     

<Emphasis Type="Italic">Drosophila</Emphasis> pupation behavior in the wild

We investigated pupa distributions of D. simulans, D. buzzatii, D. melanogaster, D. immigrans and D. hydei on a number of natural breeding sites. Pupae of all five species showed aggregated distributions, which prompted us to examine these aggregations in a more detail for two species that commonly co-occur in breeding sites, D. simulans and D. buzzatii. We found that pupae of both species tend to be aggregated in conspecific clusters. Subsequent experiments revealed that both species are attracted to the odors of other larvae, though only D. buzzatii differentiated between conspecifics and heterospecifics (they preferred conspecific). Furthermore, third instar larvae of both species preferred more alkaline substrates. Altogether, our results demonstrate that Drosophila species form conspecific pupa aggregations in natural breeding sites, and that pupation site selection depends on interactions among conspecific and heterospecific larvae and on chemical characteristics of the breeding sites.     

Transposon display supports transpositional activity of <Emphasis Type="Italic">P</Emphasis> elements in species of the <Emphasis Type="Italic">saltans</Emphasis> group of <Emphasis Type="Italic">Drosophila</Emphasis>

Mobilization of two P element subfamilies (canonical and O-type) from Drosophila sturtevanti and D. saltans was evaluated for copy number and transposition activity using the transposon display (TD) technique. Pairwise distances between strains regarding the insertion polymorphism profile were estimated. Amplification of the P element based on copy number estimates was highly variable among the strains (D. sturtevanti, canonical 20.11, O-type 9.00; D. saltans, canonical 16.4, O-type 12.60 insertions, on average). The larger values obtained by TD compared to our previous data by Southern blotting support the higher sensitivity of TD over Southern analysis for estimating transposable element copy numbers. The higher numbers of the canonical P element and the greater divergence in its distribution within the genome of D. sturtevanti (24.8%) compared to the O-type (16.7%), as well as the greater divergence in the distribution of the canonical P element, between the D. sturtevanti (24.8%) and the D. saltans (18.3%) strains, suggest that the canonical element occupies more sites within the D. sturtevanti genome, most probably due to recent transposition activity. These data corroborate the hypothesis that the O-type is the oldest subfamily of P elements in the saltans group and suggest that the canonical P element is or has been transpositionally active until more recently in D. sturtevanti.     

Polymorphism of canonical and noncanonical <Emphasis Type="Italic">gypsy</Emphasis> sequences in different species of <Emphasis Type="Italic">Drosophila </Emphasis><Emphasis Type="Italic">melanogaster</Emphasis> subgroup: possible evolutionary relations

Mobile genetic elements constitute a substantial part of eukaryotic genome and play an important role in its organization and functioning. Co-evolution of retrotransposons and their hosts resulted in the establishment of control systems employing mechanisms of RNA interference that seem to be impossible to evade. However, “active” copies of endogenous retrovirus gypsy escape cellular control in some cases, while its evolutionary elder “inactive” variants do not. To clarify the evolutionary relationship between “active” and “inactive” gypsy we combined two approaches: the analysis of gypsy sequences, isolated from G32 Drosophila melanogaster strain and from different Drosophila species of the melanogaster subgroup, as well as the study of databases, available on the Internet. No signs of “intermediate” (between “active” and “inactive”) gypsy form were found in GenBank, and four full-size G32 gypsy copies demonstrated a convergence that presumably involves gene conversion. No “active” gypsy were revealed among PCR generated gypsy ORF3 sequences from the various Drosophila species indicating that “active” gypsy appeared in some population of D. melanogaster and then started to spread out. Analysis of sequences flanking gypsy variants in G32 revealed their predominantly heterochromatic location. Discrepancy between the structure of actual gypsy sites in G32 and corresponding sequences in database might indicate significant inter-strain heterochromatin diversity. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Expression of <Emphasis Type="Italic">recA</Emphasis> gene of <Emphasis Type="Italic">Deinococcus radiodurans</Emphasis> in <Emphasis Type="Italic">Escherichia coli</Emphasis> cells

Plasmids pKS5 and pKSrec30 carrying normal and mutant alleles of the Deinococcus recA gene controlled by the lactose promoter slightly increase radioresistance of Escherichia coli cells with mutations in genes recA and ssb. The RecA protein of D. radiodurans is expressed in E. coli cells, and its synthesis can be supplementary induced. The radioprotective effect of the xenologic protein does not exceed 1.5 fold and yields essentially to the contribution of plasmid pUC19-recA1.1 harboring the E. coli recA ⁺ gene in the recovery of resistance of the ΔrecA deletion mutant. These data suggest that the expression of D. radiodurans recA gene in E. coli cells does not complement mutations at gene recA in the chromosome possibly due to structural and functional peculiarities of the D. radiodurans RecA protein.     

A PCR survey of <Emphasis Type="Italic">Hox</Emphasis> genes in the myzostomid <Emphasis Type="Italic">Myzostoma cirriferum</Emphasis>

Using degenerate primers, we were able to identify seven Hox genes for the myzostomid Myzostoma cirriferum. The recovered fragments belong to anterior class (Mci_lab, Mci_pb), central class (Mci_Dfd, Mci_Lox5, Mci_Antp, Mci_Lox4), and posterior class (Mci_Post2) paralog groups. Orthology assignment was verified by phylogenetic analyses and presence of diagnostic regions in the homeodomain as well as flanking regions. The presence of Lox5, Lox4, and Post2 supports the inclusion of Myzostomida within Lophotrochozoa. We found signature residues within flanking regions of Lox5, which are also found in annelids, but not in Platyhelminthes. As such the available Hox genes data of myzostomids support an annelid relationship. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Climatic adaptation of chromosomal inversions in <Emphasis Type="Italic">Drosophila subobscura</Emphasis>

Drosophila subobscura is a species with a rich chromosomal polymorphism which is adaptive to different climatic conditions. Five samples of the Font Groga population (Barcelona, Spain) were sampled in autumn during 5 consecutive years (2011–2015) to obtain their inversion chromosomal polymorphism, and climatic data of several meteorological variables were also collected. The aim was to analyze the adaptive potential of inversions with regard to climatic variables, being the most relevant: mean temperature (T_mean), maximum temperature (T_max), minimum temperature (T_min), humidity (Hm) and rainfall (Rf). As expected, no significant variation in inversion frequencies were detected over this short period of time. However, from a climatic point of view it was possible to differentiate ‘warm’ and ‘dry’ from ‘cold’ and ‘humid’ samples. The joint study of maximum (T_max) and minimum (T_min) temperatures was a key element to understand the effect on adaptation of many inversions. It was also observed that temperature had to be considered in conjunction with humidity and rainfall. All these factors would condition the biota of D. subobscura habitat, and chromosomal inversions could provide an adaptive response to it.     

Manipulating the <Emphasis Type="Italic">Caenorhabditis elegans</Emphasis> genome using <Emphasis Type="Italic">mariner</Emphasis> transposons

Tc1, one of the founding members of the Tc1/mariner transposon superfamily, was identified in the nematode Caenorhabditis elegans more than 25 years ago. Over the years, Tc1 and other endogenous mariner transposons became valuable tools for mutagenesis and targeted gene inactivation in C. elegans. However, transposition is naturally repressed in the C. elegans germline by an RNAi-like mechanism, necessitating the use of mutant strains in which transposition was globally derepressed, which causes drawbacks such as uncontrolled proliferation of the transposons in the genome and accumulation of background mutations. The more recent mobilization of the Drosophila mariner transposon Mos1 in the C. elegans germline circumvented the problems inherent to endogenous transposons. Mos1 transposition strictly depends on the expression of the Mos transposase, which can be controlled in the germline using inducible promoters. First, Mos1 can be used for insertional mutagenesis. The mobilization of Mos1 copies present on an extrachromosomal array results in the generation of a small number of Mos1 genomic insertions that can be rapidly cloned by inverse PCR. Second, Mos1 insertions can be used for genome engineering. Triggering the excision of a genomic Mos1 insertion causes a chromosomal break, which can be repaired by transgene-instructed gene conversion. This process is used to introduce specific changes in a given gene, such as point mutations, deletions or insertions of a tag, and to create single-copy transgenes.     

Patterns of DNA-Sequence Divergence Between <Emphasis Type="Italic">Drosophila miranda</Emphasis> and <Emphasis Type="Italic">D. pseudoobscura</Emphasis>

Contrary to the classical view, a large amount of non-coding DNA seems to be selectively constrained in Drosophila and other species. Here, using Drosophila miranda BAC sequences and the Drosophila pseudoobscura genome sequence, we aligned coding and non-coding sequences between D. pseudoobscura and D. miranda, and investigated their patterns of evolution. We found two patterns that have previously been observed in comparisons between Drosophila melanogaster and its relatives. First, there is a negative correlation between intron divergence and intron length, suggesting that longer non-coding sequences may contain more regulatory elements than shorter sequences. Our other main finding is a negative correlation between the rate of non-synonymous substitutions (d _N) and codon usage bias (F _op), showing that fast-evolving genes have a lower codon usage bias, consistent with strong positive selection interfering with weak selection for codon usage.     

Association Between the <Emphasis Type="Italic">cfx</Emphasis>A Gene and Transposon Tn<Emphasis Type="Italic">4555</Emphasis> in <Emphasis Type="Italic">Bacteroides distasonis</Emphasis> Strains and Other <Emphasis Type="Italic">Bacteroides</Emphasis> Species

The Bacteroides genus, the most prevalent anaerobic bacteria of the intestinal tract, carries a plethora of the mobile elements, such as plasmids and conjugative and mobilizable transposons, which are probably responsible for the spreading of resistance genes. Production of β-lactamases is the most important resistance mechanism including cephalosporin resistance to β-lactam agents in species of the Bacteroides fragilis group. In our previous study, the cfxA gene was detected in B. distasonis species, which encodes a clinically significant broad-spectrum β-lactamase responsible for widespread resistance to cefoxitin and other β-lactams. Such gene has been associated with the mobilizable transposon Tn4555. Therefore, the aim of this study was to detect the association between the cfxA gene and the presence of transposon Tn4555 in 53 Bacteroides strains isolated in Rio de Janeiro, Brazil, by PCR assay. The cfxA gene was detected in 11 strains and the Tn4555 in 15. The transposon sequence revealed similarities of approximately 96% with the B. vulgatus sequence which has been deposited in GenBank. Hybridization assay was performed in attempt to detect the cfxA gene in the transposon. It was possible to associate the cfxA gene in 11 of 15 strains that harbored Tn4555. Among such strains, 9 presented the cfxA gene as well as Tn4555, but in 2 strains the cfxA gene was not detected by PCR assay. Our results confirm the involvement of Tn4555 in spreading the cfxA gene in Bacteroides species.     

Population dynamics of a maternally-transmitted <Emphasis Type="Italic">Spiroplasma</Emphasis> infection in <Emphasis Type="Italic">Drosophila hydei</Emphasis>

A maternally-inherited spiroplasma endosymbiont of Drosophila hydei does not exert apparent phenotypes on both sexes of its host and is prevalent in natural populations of D. hydei. Our previous experiments using a laboratory stock of D. hydei revealed that low temperatures (such as 15°C and 18°C) dramatically lower the vertical transmission rates of this spiroplasma. Therefore, we hypothesized that, in temperate regions, the infection frequencies may decrease in cool seasons but increase in the summer season. To clarify the temporal population dynamics of the spiroplasma infection, D. hydei were collected from two Japanese populations in 2006–2008 from May to early August, representing the only period when a number of D. hydei are collectable in Japan, and examined for spiroplasma infection. Within each year, the frequency of spiroplasma infection fluctuated considerably in both populations. Consistent with our hypothesis, the infection frequency showed an increasing trend in both populations in 2007. However, the data in 2006 and 2008 did not show consistent patterns of increase. The population dynamics of spiroplasma infection may be affected but not critically determined by temperature. Moreover, despite the fluctuation within each year, the infection frequencies seemed to be stable across the years. The frequencies of spiroplasma infection in D. hydei populations may be stabilized by multiple factors. One of these factors may involve a context-dependent positive effect of spiroplasma on the fitness of D. hydei, as was recently observed in laboratory experiments.