Interpopulation variation of transposable elements of the hAT superfamily in Drosophila willistoni (Diptera: Drosophilidae): in-situ approach

Abstract

Transposable elements are abundant and dynamic part of the genome, influencing organisms in different ways through their presence or mobilization, or by acting directly on pre- and post-transcriptional regulatory regions. We compared and evaluated the presence, structure, and copy number of three hAT superfamily transposons (hobo, BuT2, and mar) in five strains of Drosophila willistoni species. These D. willistoni strains are of different geographical origins, sampled across the north-south occurrence of this species. We used sequenced clones of the hAT elements in fluorescence in-situ hybridizations in the polytene chromosomes of three strains of D. willistoni. We also analyzed the structural characteristics and number of copies of these hAT elements in the 10 currently available sequenced genomes of the willistoni group. We found that hobo, BuT2, and mar were widely distributed in D. willistoni polytene chromosomes and sequenced genomes of the willistoni group, except for mar, which is restricted to the subgroup willistoni. Furthermore, the elements hobo, BuT2, and mar have different evolutionary histories. The transposon differences among D. willistoni strains, such as variation in the number, structure, and chromosomal distribution of hAT transposons, could reflect the genomic and chromosomal plasticity of D. willistoni species in adapting to highly variable environments.

Figure 1 -. Geographical origins of the Drosophila willistoni strains analyzed in silico and in situ, and information about hAT TE copies. Lines indicate the approximate geographical distributions of the three subspecies of Drosophila willistoni (Mardiros et al., 2016). The numbers of TE copies in polytene chromosomes were measured by ImageJ software (Schneider et al., 2012) and visually. The ordinal number represents stronger signals, and increasing from + to +++ indicate the relative strength of intensity of signals on chromosome arms, as detected visually on polytene chromosome arms. In the table: - indicates absence of information; c and nc indicate presence and absence of signals on the chromocenter, respectively; w indicates weak signals; +,++ and +++, increasing from + to +++ indicate the relative strength of intensity of signals detected visually on polytene chromosomes by FISH.

Figure 2 -. FISH in polytene chromosomes of Drosophila willistoni strains: (A-C) D. willistoni-Gd-H4-1; (D-F) D. willistoni-WIP-4; and (G-I) D. willistoni-SG12.00. The probes used are indicated in the lower right corner and the strains in the lower left corner of the images. Chromosomes were counterstained with DAPI (blue) and transposable element probes were labeled with Cy3 (red). Scale bar=10 µm.

Figure 3 -. Information on species and evolutionary relationships of sequenced genomes of the willistoni group. Schematic evolutionary relationships among species of the willistoni group are based on Finet et al., (2021). (-) Absence; (+) presence; State = Structural characteristics of TE; CP = Complete or Partially complete copies; DR = Degenerate or Relic copies; MITE = miniature inverted-repeat transposable elements.

Figure 4 -. Schematic representation of reconstructed hobo, BuT2, and mar copies in the willistoni group. (A) hobo: all sequences are represented; (B) BuT2: all sequences are represented and transposase is formed by 5 exons, indicated by descending ordinal numbers; (C) mar : mar-MITE and degenerate sequences in D. willistoni-Gd-H4-1, D. willistoni-L17, D. willistoni-00, D. paulistorum-L06, D. paulistorum-L12, and D. equinoxialis were grouped. Regions of terminal inverted repeats shown inside black block, transposase coding region inside red line, and probes used in FISH experiments inside pink block. Only indels and deletions of nucleotides with more than 10 bp are represented.

Figure 5 -. Phylogenetic relationships of hobo copies in the willistoni group. Unrooted Bayesian tree (GTR+G) based on nucleotide sequences. Node supports are shown by posterior probability. Drosophila melanogaster hobo canonical sequence is shown in black; the hobo_clone was used in the FISH experiments. Different strains and species are indicated in different colors, as shown in the legend. Further information on hobo sequences is available in Table S3.

Figure 6 -. Phylogenetic relationships of the BuT2 copies in the willistoni group. Unrooted Bayesian tree (HKY+G) based on nucleotide sequences. Node supports are shown by posterior probability. Drosophila buzzatti BuT2 canonical sequence is shown in black; the BuT2_clone was used in the FISH experiments. Different strains and species are indicated in different colors, as shown in the legend. Further information about BuT2 sequences is available in Table S5.

Figure 7 -. Phylogenetic relationships of the mar copies in the willistoni subgroup. A: Bayesian tree of partially complete mar copies in the sequenced genomes of D. willistoni strains, D. equinoxialis, D. tropicalis, and D. insularis. Very degenerate copies of D. tropicalis and D. insularis were excluded from this analysis. B: Bayesian tree of mar partially complete, MITEs and relic copies in the sequenced genomes of the willistoni subgroup. This tree shows partially complete and relic copies used in A, and representative copies of mar MITES and degenerate copies in the D. willistoni strains, D. paulistorum strains, and D. equinoxialis genomes. AF518731_mar is the canonical mar-MITE of D. willistoni; mar_trop was used in the FISH experiments. Three different clades are indicated in boxes I, II, and III. Different strains and species are indicated in different colors, as shown in the legend. Degenerate sequences and MITEs are indicated by asterisks. Further information about mar sequences is available in Table S7.