We worked on fly samples collected from several geographically-distinct, natural populations (confer each Method for natural populations investigated). These populations were maintained in the laboratory as isofemale lines or small mass cultures with around 50 pairs in each generation.
In situ hybridization
Polytene chromosomes from salivary glands of third-instar female larvae were prepared and treated with nick-translated, biotinylated DNA probes, as previously described . Insertion sites were visible as brown bands resulting from a dye-coupled reaction with peroxidase substrate and diaminobenzidine. The insertion site numbers of the TE(s) were determined on all the long chromosomes arms (X, 2L, 2R, 3L, 3R), and were summed to give the total number of labeled sites per diploid genome. We did not take into account the insertions located in pericentromeric regions 20, 40, 41, 80, and 81, because TE site number estimations in these regions are difficult and not reliable for all chromosomes or all squashes. We used a probe (1278 bp) from helena of D. sechellia (AF012044). The following populations of D. melanogaster were investigated: Portugal (Chicharo), Saudi Arabia, Congo (Brazzaville), Reunion Island, Argentina (Virasoro), Bolivia, China (Canton), Vietnam, and Iso line. The D. simulans populations analyzed were from France (Valence), Russia (Moscow), Kenya (Makindu), Zimbabwe, Reunion Island, Australia (Eden, Cann River and Canberra), French Polynesia (Papeete), New Caledonia (Amieu).
DNA was extracted from one or five adult females by a standard phenol-chlorophorm-salt method with proteinase K digestion. The D. melanogaster populations analyzed were from Bolivia, Congo (Brazzaville), China (Canton), Portugal (Chicharo), Reunion Island, Saudi Arabia, Argentina (Virasoro), Vietnam, and ISO line. The D. simulans populations analyzed were from New Caledonia (Amieu), Australia (Eden, Cann River and Canberra), France (Valence), French Polynesia (Papeete), Russia (Moscow), Kenya (Makindu), Zimbabwe, and Reunion Island. The DNA was cut using the Hind III enzyme, which has no restriction site within the helena sequence, and therefore allowed us to estimate the number of complete helena copies. Electrophoresis of a 0.8% agarose gel containing digested DNA was carried out for 17 h. The DNA was denatured (NaOH 0.5 M), and then transferred overnight to a Hybond-N+ nylon membrane. Pre-hybridization and hybridization were carried out at 67°C using a Denhardt 5× solution. The probe used for hybridization (AF012044) was radiolabeled with 32P, using a random procedure from Amersham.
Amplification of ORF1 and ORF2
DNA was extracted from single flies by a standard phenol-chlorophorm method. The following populations of D. melanogaster were investigated: France (Valence and Saint Cyprien), Portugal (Chicharo), Saudi Arabia, Senegal, Congo (Brazzaville), Reunion Island, Guadeloupe, Argentina (Virasoro), Bolivia, and China (Canton). The D. simulans populations analyzed were from France (Valence), Russia (Moscow), Egypt (Tanta), Congo (Brazzaville), Kenya (Meru, Kwalé and Makindu), Zimbabwe, Tanzania (Arusha), Puerto Rico, Japan, Australia (Eden, Cann River and Canberra), French Polynesia (Papeete), Saint Martin, Hawaii, New Caledonia (Amieu), and Portugal (Madeira).
PCR was run using 1 μg DNA with the two following primers – ORF1: H1for (285 5' AAC TGT AAA ATG GAT ACG AAC A 3' 306), H1rev (1808 5' GCC ACT TCA TAA ATT GTT CC 3' 1827). – ORF2: Hel2F (2325 5' CCG GGC TGG GCG ATA TGG 3' 2342), Hel2R (4548 CGT ACA TAC CAG GGG CAG TTG G 3' 4569). PCR was run in 30 cycles with annealing temperatures of 57°C (ORF1) and 56°C (ORF2). We used Euroblue taq from Eurobio. DNA amplified fragments were purified and cloned on competent bacteria (Qiagen kits). Four primers were used for sequencing: M13 forward and reverse; Seq1 (5' CTC TTC CTT CAT TTG GTA CG 3') and Seq2 (5' AAG GGG AAA CAG TGA GAA TA 3') for the complete ORF1; Seq3F (5' TTA GAC CAT GCT CTC GGT TA 3') and Seq3R (5' TGT CAA TTC CTG GAG CTT TA 3') for a fragment of ORF2. Sequencing was performed by Genome Express. Accession numbers (Genbank) from EU168807 to EU168844 correspond to ORF1 fragments. Accession numbers (Genbank) from EU170377 to EU170431 correspond to ORF2 fragments.
Total RNA was extracted from four adult females, four adult males, 10 ovaries and 10 testes from D. simulans populations (France (Valence), Congo (Brazzaville), Kenya (Makindu), Zimbabwe, Australia (Canberra), New Caledonia (Amieu), Portugal (Madeira), United States (San Antonio, San Diego, Arena, SW3)) with the RNeasy protect mini kit from Qiagen. RNA extracts were treated with the Ambion's DNA-free kit. ThermoScript RT-PCR system from Invitrogen was used to synthesize four different cDNA pools (55°C for 90 min and 85°C for 5 min): a control reaction with no retrotranscriptase to test DNA contamination, a pool of total cDNA synthesized with oligo-dt primers, two specific cDNA pools obtained with H1R (ORF1) and Hel2R (ORF2), respectively, corresponding to helena transcripts. All four cDNA pools were tested for the presence of actin cDNA (house keeping gene) by PCR with Act5cfw (5'ATGTGACGAAGAAGTTG3') and Act5cRv (5'TTAGAAGCACTTGCGGTGCA3') primers. Oligo-dt and specific helena cDNA pools were analyzed by PCR using ORF1 and ORF2 specific primers (H1R/H1F and Hel2R/Hel2F).
Total RNA was extracted from adult females or embryos from several D. simulans populations (Valence, Makindu, Amieu, Brazzaville) with the RNeasy protect mini kit from Qiagen. Total RNA extracts were treated with the Ambion's DNA-free kit. Electrophoresis (MOPS, formaldehyde gel) was run for 3 h after RNA denaturing. After washing (water and NaOH, 75 mM) RNA was passively transferred to a nylon membrane, and cross linked for 2 hours at 64°C. Blots were pre-hybridized in hybridization buffer, then hybridized overnight at 42°C in hybridization buffer containing a 32P-labeled helena cDNA probe. The radiolabeled cDNA probe was prepared using a Megaprime DNA Labeling Kit according to the manufacturer's protocol (Cat # RPN 1607; Amersham Biosciences, Little Chalfont, Buckinghamshire, England). Following hybridization, blots were washed in 2 × SSC/0.1% SDS at 42°C and then exposed to X-ray film (KODAK).
Identification of helena copies in the complete genomes
We retrieved the sequences of the chromosome arms 2L, 2R, 3L, 3R, 4, X and the unassigned part (named U) corresponding to the first release of the mosaic assembly of the genome of D. simulans available at the ftp site of the Genome Sequencing Center at the Washington University Medical School . This mosaic assembly corresponds to different strains of D. simulans. We also used the sequenced genome of D. melanogaster . We will refer to the helena copies found in the genomes according to the chromosome name and the start position of the copy (for example chr2L_133500 corresponds to a copy found on the 2L chromosome, and it starts at position 133500).
The helena element was only found in the databases as fragments of the reverse transcriptase (RT). We retrieved the longest sequences from D. yakub a (accession number in Genbank AF012049), D. melanogaster (AF012030) and D. virilis (U26847) to build a chimeric, 1532-bp sequence. Using this chimeric element we searched for copies in the D. simulans sequenced genome using blastn . The reconstructed helena sequence (ID Helena_DS) is available in Repbase . Only matches with an e-value of less than 10e-10 have been retained, and any separated by distances of less than 300 bp have been merged. As the query used corresponds to a small portion of the ORF2, in order to search for longer sequences of helena, we retrieved the matches after adding 5000 bp around their positions. We then performed multiple alignments of these sequences using clustalw  in order to detect the longest copies. By this procedure, we identified a sequence on the chromosome 3R that was the longest of the matches detected. The prediction of potentially coding parts was made using the ORF finder program available on the NCBI web site , and this allowed us to identify two ORFs. It was not possible to use the presence of target site duplication to determine the exact position of the beginning of the sequence, because the copy was surrounded by unidentified bases, and so we performed a blast search in the draft sequence of D. sechellia, the closest relative to D. simulans. This allowed us to find a homologous copy, and to identify the beginning of the complete copy of helena. Once this copy had been identified, it was used as a query to perform blast searches in the D. simulans and D. melanogaster genomes to determine the helena copy populations.
The computation of the percentage identity was performed using the DNADIST program in the PHYLIP package . We used the sequence editor Seaview  to visualize the sequences and the alignments. Splice sites and transcription binding sites were predicted by the Softberry tools  and Genomatix ; PEPcoil ( allowed us to find the coiled coil domain in ORF 1. Conserved domains in both ORF1 and ORF2 were predicted with the "Conserved domain search" tool from NCBI. Sequenced copies were aligned with T_coffee . Phylogenetic analysis were made using maximum likelihood with HKY substitution model implemented in PhyML . The reconstruction was performed on the cloned DNA sequences of the ORF1 and ORF2 region from the different populations of D. simulans, and from some sequences detected in the sequenced genome (we eliminated sequences that were too short relative to the global length of the alignment).
Age was estimated using the Bowen and McDonald method  with the formula Age = K/(2r), where K is the divergence between the two copies calculated from the Kimura two-parameter distance via DNAdist, and r is the synonymous substitution rate per site per million years in D. melanogaster (r = 0.016 from Li ). It is important to note that the age of helena copies is underestimated due to the lack of knowledge about conversion and substitution rates in D. melanogaster genome, and is also unreliable when applied to old and highly diverged copies.