New Results

Rapid evolutionary diversification of the flamenco locus across simulans clade Drosophila species

Sarah Signor, Jeffrey Vedanayagam, Bernard Y. Kim, Filip Wierzbicki, Robert Kofler, Eric C. Lai
doi: https://doi.org/10.1101/2022.09.29.510127

Abstract

Effective suppression of transposable elements (TEs) is paramount to maintain genomic integrity and organismal fitness. In D. melanogaster, flamenco is a master suppressor of TEs, preventing their movement from somatic ovarian support cells to the germline. It is transcribed by Pol II as a long (100s of kb), single-stranded, primary transcript, that is metabolized into Piwi-interacting RNAs (piRNAs) that target active TEs via antisense complementarity. flamenco is thought to operate as a trap, owing to its high content of recent horizontally transferred TEs that are enriched in antisense orientation. Using newly-generated long read genome data, which is critical for accurate assembly of repetitive sequences, we find that flamenco has undergone radical transformations in sequence content and even copy number across simulans clade Drosophilid species. D. simulans flamenco has duplicated and diverged, and neither copy exhibits synteny with D. melanogaster beyond the core promoter. Moreover, flamenco organization is highly variable across D. simulans individuals. Next, we find that D. simulans and D. mauritiana flamenco display signatures of a dual-stranded cluster, with ping-pong signals in the testis and/or embryo. This is accompanied by increased copy numbers of germline TEs, consistent with these regions operating as functional dual stranded clusters. Overall, the physical and functional diversity of flamenco orthologs is testament to the extremely dynamic consequences of TE arms races on genome organization, not only amongst highly related species, but even amongst individuals.

Introduction

Drosophila gonads exemplify two important fronts in the conflict between transposable elements (TEs) and the host – the germline (which directly generates gametes), and somatic support cells (from which TEs can invade the germline) (1, 2). The strategies by which TEs are suppressed in these settings are distinct (3), but share their utilization of piwi-interacting RNAs (piRNAs). These are ~24-32 nt RNAs that are bound by the PIWI subclass of Argonaute proteins, and guide them and associated cofactors to targets for transcriptional and/or post-transcriptional silencing (4–7).

Mature piRNAs are processed from non-coding piRNA cluster transcripts, which derive from genomic regions that are densely populated with TE sequences (7–9). However, the mechanisms of piRNA biogenesis differ between gonadal cell types. In the germline, piRNA clusters are transcribed from both DNA strands through non-canonical Pol II activity (6, 10–12), which is initiated by chromatin marks rather than specific core promoter motifs. Moreover, co-transcriptional processes such as splicing and polyadenylation are suppressed within dual strand piRNA clusters (13, 14). On the other hand, in ovarian somatic support cells, piRNA clusters are transcribed from a typical promoter as a single stranded transcript, which can be alternatively spliced as with protein-coding mRNAs (15–18). These rules derive in large part from the study of model piRNA clusters (i.e. the germline 42AB and somatic flamenco piRNA clusters). For both types, their capacity to repress invading TEs is thought to result from random integration of new transposons into the cluster (19). As such, piRNA clusters are adaptive loci that play central roles in the conflict between hosts and TEs.

The location and activity of germline piRNA clusters are stochastic and evolutionarily dynamic, as there are many copies of TE families in different locations that may produce piRNAs (9, 20). By contrast, somatic piRNA clusters are not redundant and a single insertion of a TE into a somatic piRNA cluster should be sufficient to prevent that TE from further transposition (1, 18). Thus, flamenco should contain only one copy per TE family (18), which is true in the flamenco locus of D. melanogaster (18). flamenco is also the only piRNA cluster which produces a phenotypic effect when altered, as germline clusters can be deleted with no consequences (9).

flamenco has been a favored model for understanding the piRNA pathway since the discovery of piRNA mediated silencing of transposable elements (6). flamenco spans >180 kb of repetitive sequences located in β-heterochromatin of the X chromosome (21). Of note, flamenco was initially identified, prior to the formal recognition of piRNAs, via transposon insertions that de-repress gypsy, ZAM, and Idefix class elements (21–25). These mutant alleles disrupt the flamenco promoter, and consequently abrogate transcription and piRNA production from this locus. By contrast, the recent deletion of multiple model germline piRNA clusters, which eliminate the biogenesis of a bulk of cognate piRNAs, did not de-repress their cognate TEs (9). Thus, the analysis of flamenco evolution is presumably more consequential for TE dynamics. Analysis of flamenco in various strains of D. melanogaster supports that this locus traps horizontally derived TEs to achieve silencing of newly invaded TEs (18). The flamenco locus exhibits synteny across the D. melanogaster sub-group (26); however, the sequence composition of flamenco outside D. melanogaster has not been well-characterized (27).

In this study, we compare the flamenco locus across 10 strains of simulans-clade species, namely D. simulans, D. mauritiana, and D. sechellia. Analysis of piRNAs from ovaries of five genotypes of D. simulans found that flamenco is duplicated in D. simulans. This duplication is old enough that there is no sequence synteny across copies, even though their core promoter regions and the adjacent dip1 gene duplications are conserved. flamenco has also been colonized by abundant (>40) copies of R1, a TE that was thought to insert only at ribosomal genes, and to evolve at the same rate as nuclear genes (28). Furthermore, between different genotypes, up to 63% of TE insertions are not shared within any given copy of flamenco. Despite this, several full length TEs are shared between all genotypes in a similar sequence context. This incredible diversity at the flamenco locus, even within a single species, suggests there may be considerable variation in its ability to suppress transposable elements across individuals.

Cross-species comparisons further indicate that functions of flamenco have diversified. Data from D. sechellia and D. melanogaster conform with the current understanding of flamenco as a uni-strand cluster. However, we find evidence that D. simulans and D. mauritiana flamenco can act as a dual strand cluster in testis (D. mauritiana) and embryos (D. mauritiana and D. simulans), yielding piRNAs from both strands with a ping pong signal. Overall, we infer that the rapid evolution of flamenco alleles across individuals and species reflects highly adaptive functions and dynamic biogenesis capacities.

Materials and Methods

Fly strains

The four D. simulans lines SZ232, SZ45, SZ244, and SZ129 were collected in California from the Zuma Organic Orchard in Los Angeles, CA on two consecutive weekends of February 2012 (29–33). LNP-15-062 was collected in Zambia at the Luwangwa National Park by D. Matute and provided to us by J. Saltz (J. Saltz pers. comm., (34, 35)). MD251, MD242, NS137, and NS40 were collected in Madagascar and Kenya (respectively) and are described in (36). The D. simulans strain wxD1 was originally collected by M. Green, likely in California, but its provenance has been lost (pers. comm. Jerry Coyne). D. mauritiana (w12) and D. sechellia (Rob3c/Tucson 14021-0248.25) are described in (37).

Long read DNA sequencing and assembly

MD242, four SZ lines and LNP-15-062 were sequenced on a MinION platform at North Dakota State University (Oxford Nanopore Technologies (ONT), Oxford, GB), with base-calling using guppy (v4.4.2). MD242, the four SZ lines, and LNP-15-062 were assembled with Canu (v2.1) (38) and two rounds of polishing with Racon (v1.4.3) (39). The CA strains were additionally polished with short reads using Pilon (v1.23) (40)(SRR3585779, SRR3585440, SRR3585480, SRR3585391) (29). The first wxD1-1 assembly is described here (41). MD251, NS137, NS40 and wxD1-2 were sequenced on a MinION platform at Stanford University. They were assembled with Flye (42), and polished with a round of Medaka followed by a round of pilon (40). Following this contaminants were removed with blobtools (https://zenodo.org/record/845347, (43)), soft masked with RepeatModeler and Repeatmasker (44, 45), then aligned to the wxD1 as a reference with Progressive Cactus (46). The assemblies were finished with reference based scaffolding using Ragout (47). D. mauritiana and D. sechellia were sequenced with PacBio and assembled with FALCON using default parameters (https://github.com/PacificBiosciences/FALCON)(37).The D. melanogaster assembly is described here (48). A summary of the assembly statistics is available in Supplementary Table 1. The quality of cluster assembly was evaluated using the coverage and soft clip quality as described in (20, 49) (Supplementary File 1).

Short read sequencing and mapping

Short read sequencing was performed by Beijing Genomics Institute on approximately 50 dissected ovaries from adult female flies (SZ45, SZ129, SZ232, SZ244, LNP-15-062). Short read libraries from 0-2 hour embryos were prepared from D. melanogaster, wxD1-2, D. sechellia, and D. mauritiana (SRAXXX) (50). Small RNA from testis is described in (51, 52). D. melanogaster OSC small RNA libraries were downloaded from the SRA (SRR11999160). Libraries were filtered for adapter contamination and short reads between 23-29 bp were retained for mapping with fastp (53). The RNA was then mapped to their respective genomes using bowtie (v1.2.3) and the following parameters (-q -v 1 -p 1 -S -a - m 50 --best --strata) (54, 55). The resulting bam files were processed using samtools (56). To obtain unique reads the bam files were filtered for reads with 1 mapping position. To obtain counts files with weighted mapping the bam files were processed using Rsubreads and the featureCounts function (57).

Defining and annotating piRNA clusters

piRNA clusters were initially defined using proTRAC (58). piRNA clusters were predicted with a minimum cluster size of 1 kb (option “-clsize 1000”), a p-value for minimum read density of 0.07 (option “-pdens 0.07”), a minimum fraction of normalized reads that have 1T (1U) or 10A of 0.33 (option “-1Tor10A 0.33”) and rejecting loci if the top 1% of reads account for more than 90% of the normalized piRNA cluster read counts (option “-distr 1-90”), and a minimal fraction of hits on the main strand of 0.25 (option “-clstrand 0.25”). Note that this ties the piRNA clusters to their function such that participation in the ping pong pathway can be inferred from these patterns. Clusters were annotated using RepeatMasker (v. 4.0.7) and the TE libraries described in Chakraborty et al. (2019) (41, 44). The position of flamenco was also evaluated based off of the position of the putative promoter, the dip1 gene, and the enrichment of gypsy elements (15). Fragmented annotations were merged to form TE copies with onecodetofindthemall (59). Fragmented annotations were also manually curated within flamenco, particularly because TEs not present in the reference library often have their LTRs and internal sequences classified as different elements.

Aligning the flamenco promoter region

The region around the flamenco promotor was extracted from each genotype and species with bedtools getfasta (60). Sequences were aligned with clustal-omega and converted to nexus format (61). Trees were built using a GTR substitution model and gamma distributed rate variation across sites (62). Markov chain monte carlo iterations were run until the standard deviation of split frequencies was below .01, around one million generations. The consensus trees were generated using sumt conformat=simple. The resulting trees were displayed with the R package ape (63).

Detecting ping pong signals in the small RNA data

Ping pong signals were detected using pingpongpro (64) This program detects the presence of RNA molecules that are offset by 10 nt, such that stacks of piRNA overlap by the first 10 nt from the 5’ end. These stacks are a hallmark of piRNA mediated transposon silencing. The algorithm also takes into account local coverage and the presence of an adenine at the 10th position. The output includes a z-score between 0 and 1, the higher the z-score the more differentiated the ping pong stacks are from random local stacks.

Annotating shared and unique TE insertions

To align the TE annotations of homologous piRNA clusters, we first extracted the sequences of the clusters and annotated TEs in these sequences using RepeatMasker (open-4.0.7) with a custom TE library and the parameters: -s (sensitive search), -nolow (disable masking of low complexity sequences), and -no_is (skip check for bacterial IS) (37, 65, 66). Finally, we aligned the resulting repeat annotations with Manna using the parameters -gap 0.09 (gap penalty), -mm 0.1 (mismatch penalty) -match 0.2 (match score) (20, 37). Manna can be used for aligning the annotations of the transposable elements by relying on synteny to determine insertion homology. Alignments were manually checked for inconsistencies arising from assignment to similar TEs (i.e. gypsy-3 versus gypsy-5). TEs were considered to be full length if they were present in at least 70% of their reference length and contained internal sequence as well as two LTRs if applicable.

Results

flamenco in the D. simulans clade

We identified D. simulans flamenco from several lines of evidence: piRNA cluster calls from proTRAC, its location adjacent to divergently transcribed dip1, the existence of conserved core flamenco promoter sequences, and enrichment of gypsy elements (Figure 1 & 2); Supplementary Table 2). The flamenco locus is at least 376 kb in D. simulans. This is an expansion compared with D. melanogaster, where flamenco is only 156 kb (Canton-S). In D. sechellia flamenco is 363 kb, however in D. mauritiana the locus has expanded to at least 840 kb (Supplementary Table 2). This is a large expansion, and it is possible that the entire region does not act as a region controlling somatic TEs. However, evidence that is does include uniquely mapping piRNAs that are found throughout the region and gypsy enrichment consistent with a flamenco-like locus (Supplementary Figure 1). There are no protein coding genes within the region, and while the neighboring genes on the downstream side of flamenco in D. melanogaster have moved in D. mauritiana (CG40813- CG41562 at 21.5 MB), the following group of genes beginning with CG14621 is present and flanks flamenco as it is annotated. Thus in D. melanogaster the borders of flamenco are flanked by dip1 upstream and CG40813 downstream, while in D. mauritiana they are dip1 upstream and CG14621 downstream. Between all species the flamenco promoter and surrounding region, including the dip1 gene, are alignable and conserved (Figure 1D).

Figure 1.
Figure 1.

A) The duplication of flamenco in the D. simulans. Both copies are flanked by copies of the dip1 gene and copies of the putative flamenco promoter. The top portion of the alignment shows ~ 2 kb around the promoter. SNPs are shown if they differentiate copies of flamenco within a single genotype of D. simulans. Dots do not indicate a single nucleotide, but rather a sequence region where no SNPs differentiate the two copies of flamenco within a single genotype. The lower portion illustrates the promoter region with all SNPs illustrated in D. melanogaster, D. sechellia, D. mauritiana, and D. simulans. B) A schematic of the restriction digest used to verify the duplicate of flamenco. The targeted region is a 1 kb fragment adjacent to the promotor of flamenco. Within this region the original flamenco copy does not contain a YACGTR site and is not cut by the restriction enzyme BsaAI. The duplicate of flamenco is cut into two pieces (750 bp and 250 bp). C) A gel showing the fragments of the original and duplicated copy of flamenco before and after digestion with BsaAI. Both copies of flamenco are amplified by the primers, in column two of the gel (Supplemental File 2). In column three of the gel, the original copy of flamenco is uncut (band 1), while the duplicate of flamenco forms two bands at 750 bp (band 2) and 250 bp (band 3).

Figure 2.
Figure 2.

A) Unique piRNA from the ovary and gypsy enrichment around flamenco and its duplicate in D. simulans and D. melanogaster. piRNA mapping to the entire contig that contains flamenco is shown for both species. The top of the panel shows piRNA mapping to flamenco and is split by antisense (blue) and sense (red) piRNA. The bottom panel shows the frequency of gypsy-type transposon annotations across the contig containing flamenco, counted in 100 kb windows. There is a clear enrichment of gypsy in the area of flamenco and, in D. simulans, its duplicate compared to the rest of the contig. B) The distribution of read size for small RNA mapping to flamenco. The peak is at approximately 26 bp, within the expected range for piRNA. C) The percent of TEs in flamenco in each species which are in the antisense orientation (first bar) and the percent of TEs in the antisense orientation that are also LTR class elements (second bar). D) A phylogenetic tree of the dip1 and flamenco enhancer region for D. melanogaster and the simulans clade. This region is conserved and alignable between all species. The tree was generated with Mr. Bayes (62).

Structure of the flamenco locus

D. melanogaster flamenco bears a characteristic structure, in which the majority of TEs are gypsy-class elements in the antisense orientation (79% antisense orientation, 85% of which are gypsy elements) (Figure 2C; Supplementary Table 3). In D. simulans, flamenco has been colonized by large expansions of R1 transposable element repeats such that on average the percent of antisense TEs is only 50% and the percent of the locus comprised of LTR elements is 55%. However, 76% of antisense insertions are LTR insertions, thus the underlying flamenco structure is apparent when the R1 insertions are disregarded (Figure 2C). In D. mauritiana flamenco is 71% antisense, and of those antisense elements it is 85% LTRs. Likewise in D. sechellia 78% of elements are antisense, and of those 81% are LTRs. flamenco retains the overall structure of a canonical D. melanogaster-like flamenco locus in all of these species, however in D. simulans the nature of the locus is somewhat altered by the abundant R1 insertions (Figure 2C).

flamenco is duplicated in D. simulans

In D. simulans, we unexpectedly observed that flamenco is duplicated on the X chromosome; the duplication was confirmed with PCR and a restriction digest (Figure 1, Supplementary File 2). These duplications are associated with a conserved copy of the putative flamenco enhancer as well as copies of the dip1 gene located proximal to flamenco in D. melanogaster (Figure 1, 3A). While it is unclear which copy is orthologous to D. melanogaster flamenco, all D. simulans lines bear one copy that aligns across genotypes. We refer to this copy as D. simulans flamenco, and the other copies as duplicates. Otherwise, flamenco duplicates do not align with one another and lack synteny amongst their resident TEs. Possible evolutionary scenarios are that the flamenco duplication occurred early in the simulans lineage, that the clusters evolved very rapidly, or that the duplication encompassed only the promoter region and was subsequently colonized by TEs (Figure 1A, 3A).

Figure 3.
Figure 3.

A) Divergence between copies of flamenco. Proximal is a phylogenetic tree of dip1 and the flamenco promoter region from each genome. In between dip1 and the promoter are a series of G5/INE1 repeats that are found in every genome. Overall this region is fairly conserved, with the duplicate copies all grouping together with short branch lengths (shown in pink). The original copy of flamenco is more diverse with some outliers (shown in light blue) but there is good branch support for all the deep branches of the tree. Distal is a representation of flamenco and its duplicate. R1 repeat regions are shown in blue. Full length transposable elements are labeled. There is no synteny conservation between flamenco and its duplicate. B) The proportion of insertions that are shared by one through seven genotypes (genotypes with complete flamenco assemblies). C) Divergence of flamenco within D. simulans. Labeled TEs correspond to elements which are present in a full length copy in at least one genome. If they are shared between genomes they are labeled in red, if they are unique they are black. If they are full length in one genome and degraded in other genomes they are represented by stacked dashes. If they are present in the majority of genomes but missing in one, it is represented as a missing that TE, which is agnostic to whether it is a deletion or the element was never present

The flamenco duplicate is absent in the D. simulans reference assembly, w501 (GCA_000754195.3), but present in wxD1, suggesting it was polymorphic or absent between the collection of these strains (or was not assembled). The duplicate retains the structure of flamenco, with an average of 67% of TEs in the antisense orientation, and 91% of the TEs in the antisense orientation are LTRs. The duplicate of flamenco is less impacted by R1, with some genotypes having as few as 8 R1 insertions (Figure 3C).

R1 LINE elements at the flamenco locus

R1 elements are well-known to insert into rDNA genes, are transmitted vertically, and evolve similarly to the genome background rate (28). They have also been found outside of rDNA genes, but only as fragments. R1 elements are absent from flamenco in the D. simulans reference assembly, aside from a single fragmented R1-1 element (w501). However, as mentioned, R1 elements are abundant within flamenco loci in the simulans clade. Outside of flamenco, R1 elements in D. simulans are distributed according to expectation, with full length elements occurring only within rDNA (Supplementary File 3). Within flamenco, most copies of R1 occur as tandem duplicates, creating large islands of fragmented R1 copies (Figure 3A). They are on average 3.7% diverged from the reference R1 from D. simulans. Across individual D. simulans genomes, ~99 kb of flamenco loci consists of R1 elements, i.e. 26% of their average total length. SZ45, LNP-15-062, NS40, MD251, and MD242 contain 4-7 full length copies of R1 in the sense orientation, even though all but SZ45 bear fragmented R1 copies on the antisense strand. (The SZ45 flamenco assembly is incomplete). As the antisense R1 copies are expected to suppress R1 transposition, flamenco may not suppress these elements effectively. Alternatively, it is possible that D. simulans flamenco is still mostly active in the soma, while R1 is active in the germline, and thus escapes host control by flamenco.

In D. mauritiana, flamenco harbors abundant fragments or copies of R1 (19 on the reverse strand and 20 on the forward strand), and only one large island of R1 elements. In total, D. mauritiana contains 84 kb of R1 sequence within flamenco. In D. mauritiana there are 8 full length copies of R1 at the flamenco locus, 7 in antisense, which are not obviously due to a segmental or local duplication. Finally, we find that D. sechellia flamenco lacks full length copies of R1, and it contains only 18 KB of R1 sequence (16 fragments on the reverse strand). Yet, all the copies are on the sense strand, which would not produce fragments that can suppress R1 TEs. Essentially the antisense copies of R1 in D. mauritiana should be suppressing the TE, but we see multiple full length antisense insertions, and D. sechellia has no antisense copies, but we see no evidence for recent R1 insertions. From this it would appear that whatever is controlling the transposition of R1 lies outside of flamenco.

The presence of long sense-strand R1 elements within flamenco is a departure from expectation (18, 28). There is no evidence of an rDNA gene within the flamenco locus that would explain the insertion of R1 elements there, nor is there precedence for the large expansion of R1 fragments within the locus. Furthermore, the suppression of R1 transposition does not appear to be controlled by flamenco.

piRNA production from R1

On average R1 elements within the flamenco locus of D. simulans produce more piRNA than any other TE within flamenco (Supplementary Table 6). R1 reads mapping to the forward strand constitute an average of 51% of the total piRNAs within the flamenco locus from the maternal fraction, ovary, and testis using weighted mapping. The only exception is the ovarian sample from SZ232 which is a large outlier at only 5%. However reads mapping to the reverse strand account for an average of 84% of the piRNA being produced from the strand in every genotype and tissue – maternal fraction, testis, or ovary. If unique mapping is considered instead of weighted these percentages are reduced by approximately 20%, which is to be expected given that R1 is present in many repeated copies. Production of piRNA from the reverse strand seems to be correlated with elements inserted in the sense orientation, of which the vast majority are R1 elements in D. simulans (Supplementary Figure 2). The production of large quantities of piRNA cognate to the R1 element is seemingly pointless – if R1 only inserts at rDNA genes and are vertically transmitted there is little reason to be producing the majority of piRNA in response to this element.

In D. sechellia there are very few piRNA produced from flamenco in these tissues, and there are no full length copies of R1. Likewise overall weighted piRNA production from R1 elements on either strand is 2.8-5.9% of the total mapping piRNA. In contrast in D. mauritiana there are full length R1 elements and abundant piRNA production in the maternal fraction and testis. In D. mauritiana an average of 28% of piRNAs mapping to the forward strand of flamenco are arising from R1, and 33% from the reverse strand. In D. mauritiana R1 elements make up a smaller proportion of the total elements in the sense orientation (24%), versus D. simulans (55%).

Conservation of flamenco

The dip1 gene and promoter region adjacent to each copy of flamenco are very conserved both within and between copies of flamenco (Figure 3A). The phylogenetic tree of the area suggests that we are correct in labeling the two copies as the original flamenco locus and the duplicate (Figure 3A). The original flamenco locus is more diverged amongst copies while the duplicate clusters closely together with short branch lengths (Figure 3A). The promotor region is also conserved and alignable between D. melanogaster, D. sechellia, D. mauritiana, and D. simulans (Figure 1D). However, the same is not true of the flamenco locus itself. Approximately 3 kb from the promoter flamenco diverges amongst genotypes and species and is no longer alignable by traditional sequence-based algorithms, as the TEs are essentially presence/absence polymorphisms that span multiple kb. There is no conservation of flamenco between D. melanogaster, D. simulans, D. sechellia, and D. mauritiana (Figure 4). However, within the simulans clade many of the same TEs occupy the locus, suggesting that they are the current genomic invaders in each of these species (Figure 4).

Figure 4)
Figure 4)

A. Copy number of a subset of transposable elements at flamenco. Solo LTRs are indicated by in a lighter shade at the top of the bar. The black line on each bar graph indicates a copy number of one. Values for D. simulans are the average for all genotypes with a complete flamenco assembly. Note that in D. melanogaster (green) most TEs have a low copy number. The expansion of R1 elements in the simulans clade is clearly indicated on the right hand panel with a dotted box. Many elements within flamenco are multicopy in the simulans clade. While some of this is likely due to local duplications it is clearly a different pattern than D. melanogaster. Enrichment of LTR elements on the antisense strand is clear for all species. B. Alignment of flamenco in D. melanogaster, D. simulans, D. sechellia, and D. mauritiana. There is no conserved synteny between species but there are clearly shared TEs, particularly within the simulans clade. The expansion of D. mauritiana compared to the other species is apparent.

In D. simulans the majority of full length TEs are singletons – 54% in flamenco and 64% in the duplicate. Copies that are full length in one genotype but fragmented in others are counted as shared, not singletons. Almost half of these singletons in the duplicate are due to a single genotype with a unique section of sequence, in this case MD251. Singletons are the single largest category of transposable element insertions, followed by fixed insertions. Thus even within a single population there is considerable diversity at the flamenco locus, and subsequently diversity in the ability to suppress transposable elements. For example, gypsy-29 is present in three genotypes either in flamenco or the duplicate, which would suggest that these genotypes are able to suppress this transposable element while the other genotypes are not. In contrast gypsy-3 is present in more than one full length copy in flamenco and its duplicate in every genotype but one where it is present in a single copy. There are a number of these conserved full length TEs that are present in all or nearly all genotypes, including Chimpo, gypsy-2, Tirant, and gypsy-4. In addition, the INE1 elements adjacent to the promoter are conserved.

It is notable that any full length TEs are shared across all genotypes, given that wxD1 was like collected 30-50 years prior to the others, and the collections span continents (Figure 3C). Two facts are relevant to this observation: (1) TEs were shown not to correlate with geography (67) and (2) D. simulans is more diverse within populations than between different populations (68–70). Other explanations are also plausible. Selection could be maintaining these full length TEs, wxD1 could have had introgression from other lab strains, or a combination of these explanations.

Suppression of TEs by the flamenco locus and the trap model of TE control

In D. melanogaster, it was proposed that while germline clusters may have many insertions of a single TE, the somatic ‘master regulator’ flamenco will have a single insertion of each transposon, after which they are silenced and no longer able to transpose (18).

Here, we evaluate the following lines of evidence to determine if they support the trap model of transposable element suppression. (1) How many TEs have antisense oriented multicopy elements within flamenco? (2) How many TEs have full length and fragmented insertions, suggesting the older fragments did not suppress the newer insertion? (3) How many de novo insertions of TEs in the flamenco duplicate of D. simulans are also present in the original flamenco copy?

How many TEs have antisense oriented multicopy elements within flamenco?

Due to the difficulty in classifying degraded elements accurately, for example between multiple classes of gypsy element, we will focus here on full length TEs, suggesting recent transposition. In D. melanogaster there are 7 full length elements, none of which are present in more than one antisense copy. These elements make up 27% of the flamenco locus. Full length copies of five of these elements were also reported previously for other strains of D. melanogaster (18)

In D. sechellia there are 14 full length TEs within the flamenco locus, three of which are present in multiple copies. Two of these, INE1 and 412, are likely present due to local duplication. In particular the INE1 elements flanking the promoter, are in the sense orientation, and are conserved between D. sechellia, D. mauritiana, and D. simulans. The only element present in multiple antisense copies is GTWIN. Similar to D. melanogaster these 14 elements make up 27% of the flamenco locus.

D. mauritiana contains 22 full length TEs within the flamenco locus. Four of these are present in multiple antisense full length copies – INE1, R1, Stalker-4, and Cr1a. While some of the five antisense copies of R1 likely originated from local duplications – they are in the same general region and tend to be flanked by gypsy-8, not all of them show these patterns. Furthermore, as aforementioned, there also are full length sense copies of R1 suggesting R1 is not being suppressed by flamenco. gypsy-12 and gypsy-3 have a second antisense copy within flamenco that is just below the cutoff to be considered full length – in gypsy-3 the second copy is 10% smaller, for gypsy-12 it is present but missing an LTR. Full length TEs make up 19% of the flamenco locus.

In D. simulans there are 24 full length TEs present in any of the seven complete flamenco assemblies. Six of these are present in multiple antisense copies within a single genome – INE1, Chimpo, gypsy-4, 412, Tirant, and BEL-unknown. The two Tirant copies are likely a segmental duplication as they flank an R1 repeat region. In addition, most INE1 copies are present proximal to the promoter as aforementioned. Chimpo is present in three full length copies within MD242 flamenco, with no evidence of local duplication. While there are no full length copies of R1 inserted in antisense, R1 is present in full length sense copies despite many genomes containing antisense fragments, suggesting flamenco is not suppressing R1. On average full length TEs constitute 20% of flamenco in D. simulans.

In the duplicate of flamenco in D. simulans there are 30 full length TEs present in any one of the five complete flamenco duplicate assemblies. However, none of them are multicopy in antisense. However, they are multicopy relative to the original copy of flamenco. gypsy-3, BEL-unknown, Nomad-1, Chimpo, gypsy-53A, R1, and INE1 are all multicopy with respect to the original flamenco within a given genome. Some of these may have been inherited at the time of duplication, however are full length in both copies suggesting recent transposition. In the duplicate of flamenco full length TEs occupy an average of 17% of the locus. MD251 is an exception which weights the average, with 28% of the locus, while between 10 and 15% is found for the remaining copies. Thus D. simulans and D. mauritiana overall do not meet the expectation that flamenco will contain a single insertion of any given TE.

How many TEs have full length and fragmented insertions?

Full length elements are younger insertions than fragmented insertions. If a full length element is inserted in flamenco and there are fragments in the antisense orientation elsewhere in flamenco this indicates that flamenco did not successfully suppress the transposition of this element.

In D. melanogaster two elements have fragments in antisense and a full length TE – Doc and Stalker-2. D. sechellia has 9 elements that are present as a full length TE and a fragment in antisense (including 412, GTWIN, mdg-1, and nomad) and 6 that are multicopy that are due to a solo LTR (including blood, 297, and Stalker-4). D. mauritiana has 21 elements that are present in full length and a fragment in antisense (including blood, 412, gypsy-10-13, and R1), and four elements that are multicopy due to a solo LTR (mdg-1, Idefix, and gypsy-7,10).

In D. simulans, TEs that fit this criteria in flamenco include gypsy-2, gypsy-3, gypsy-4, gypsy-5, Chimpo, 412, INE1, R1, Tirant, and Zam. 297 and Nomad-1 are present in full length copies but only multi-copy in the context of solo LTRs. In the duplicate of flamenco in D. simulans this includes gypsy-2, gypsy-3, gypsy-5, 297, Stalker-4, and R1. For example in NS40 there are 7 full length copies of R1 in the sense orientation that likely duplicated in place, as well as 12 partial copies in the antisense orientation. In the simulans clade either fragments of TEs are not sufficient to suppress transposable elements or some elements are able to transpose despite the hosts efforts to suppress them.

Is flamenco a trap for TEs entering through horizontal transfer?

High sequence similarity between TEs in different species suggests horizontal transfer (71). However, because sequence similarity can also exist due to vertical transmission we will use sequence similarity between R1 elements (inserted at rDNA genes) as a baseline for differentiating horizontal versus vertical transfer. There has never been any evidence found for horizontal transfer of R1 and it is thought to evolve at the same rate as nuclear genes in the melanogaster subgroup (18, 28). Of the full length elements present in any genome at flamenco 62% of them appear to have originated from horizontal transfer. This is similar to previous estimates for D. melanogaster in other studies (18). Transfer appears to have occurred primarily between D. melanogaster, D. sechellia, and D. willistoni. This includes some known horizontal transfer events such as Chimpo and Chouto (72), and others which have not been recorded such as gypys-29 (D. willistoni) and the Max-element (D. sechellia) (Supplementary File 4). The duplicate of flamenco is similar, with 53% of full length TEs originating from horizontal transfer. They are many of the same TEs, with a 46% overlap, thus flamenco and its duplicate are trapping many of the same TEs. Both flamenco and the duplicate the region appears to serve as a trap for TEs originating from horizontal transfer.

In D. melanogaster 85% of full length TEs appear to have arisen through horizontal transfer, which is consistent with previous estimates (18). In D. sechellia 53% of full length TEs have arisen from horizontal transfer, including some known to have moved by horizontal transfer such as GTWIN (D. melanogaster/D. erecta) (72). D. mauritiana has 68% of its full length TEs showing a closer relationship than expected by vertical descent with TEs from D. sechellia, D. melanogaster, and D. simulans. The hypothesis that flamenco serves as a trap for TEs entering the population through horizontal transfer holds throughout the simulans clade.

Flamenco piRNA is expressed in the testis and the maternal fraction

Canonically, flamenco piRNA is expressed in the somatic follicular cells of the ovary and not in the germline, and also does not produce a ping pong signal (24). It was not thought to be present in the maternal fraction of piRNAs or other tissues. However, that appears to be variable in different species (Figure 5). We examined single mapping reads in the flamenco region from testes and embryos (maternal fraction) in D. simulans, D. mauritiana, D. sechellia, and D. melanogaster. As a control we also included D. melanogaster ovarian somatic cells, where Aub and Ago3 are not expressed and therefore there should be no ping pong signals. In D. simulans and D. mauritiana flamenco is expressed bidirectionally in the maternal fraction and the testis, including ping pong signals on both strands (Figure 5A & C; Supplementary Figure 1). In D. sechellia, there is no expression of flamenco in either of these tissues. Discarding multimappers in the maternal fraction 63% (D. mauritiana) – 36% (D. simulans) of the ping pong signatures on the X with a z-score of at least 0.9 are located within flamenco (Figure 5C). In the testis the picture is more complicated – in D. mauritiana 50% of ping pong signals on the X with a z-score of at least 0.9 are located within flamenco, which amounts to a substantial ping pong signature (Supplementary Figure 1). While mapping of piRNA to both strands was observed in D. simulans testis, there is very little apparent ping pong activity (5 positions in flamenco z > 0.9; 15 potential ping pong signals on the X). In D. melanogaster, there is uni-strand expression in the maternal fraction, but it is limited to the region close to the promoter. In D. melanogaster no ping pong signals have a z-score above 0.8 in the maternal fraction or the ovarian somatic cells. There are ping pong stacks in flamenco in the testis of D. melanogaster (2% of the total on the contig), however they are limited to a single region and are not abundant enough to be strong evidence of ping pong activity.

Figure 5)
Figure 5)

A. Expression of single mapping piRNAs in the maternal fraction and testis (gray) of D. melanogaster and the simulans clade. Antisense mapping reads are shown in blue, sense in red. Libraries are RPM normalized and scaled across library type. D. sechellia has no expression of flamenco in the maternal fraction or the testis. D. melanogaster has low expression in the maternal fraction and very little ping pong activity. D. simulans and D. mauritiana show dual stranded expression in the testis and maternal fraction. B. The total number of uniquely mapping reads for each of the libraries illustrated in A. This is included to demonstrate that a low number of mapping reads does not explain the patterns seen in D. sechellia versus D. mauritiana. C. The height of 10 nt pingpong stacks at flamenco in D. melanogaster maternal fraction, testis and ovarian somatic cells is shown on the left. Below each schematic of the height of the stacks is the position of z-scores over 0.8, indicating the likelihood that this is a real ping pong signal as opposed to an artifact. Signals move from red to blue as they approach 1. In the testis a few ping pong signals reach this threshold but not enough to indicate convincingly that there is ping pong activity. On the right are the ping pong stacks and z-scores for the maternal fraction and testis in D. simulans. Only in the maternal fraction are the density of z-scores over 0.8 convincing enough to indicate an active ping pong cycle in the flamenco region. However, the presence of stacks is enriched in testis, thus this may warrant further investigation. D. mauritiana also has convincing ping pong signals in this region (Supplementary Figure 1). D. A schematic of the evolution of flamenco and its mode expression in the simulans and melanogaster clade.

In the duplicate of flamenco in the maternal fraction 15% of the ping pong signals with a z-score above 0.9 on the X are within the flamenco duplicate. The flamenco duplicate does not have a strong signal of the ping pong pathway in the testis. In addition, flamenco in these species has been colonized by full length TEs thought to be active in the germline such as blood, burdock, mdg-3, Transpac, and Bel (73, 74). blood is also present in D. melanogaster in a full length copy while there is no evidence of germline activity for flamenco in D. melanogaster, though no other putative germline TEs are present. The differences in ping pong signals between species and the presence of germline TEs in D. simulans and D. mauritiana suggests that the role of flamenco in these tissues has evolved between species.

Discussion

The piRNA pathway is the organisms primary mechanism of transposon suppression. While the piRNA pathway is conserved, the regions of the genome that produce piRNA are labile, particularly in double stranded germline piRNA clusters (9). The necessity of any single cluster for TE suppression in the germline piRNA pathway is unclear, but likely redundant (9). However, flamenco is thought to be the master regulator of the somatic support cells of the ovary, preventing gypsy elements from hopping into germline cells (18, 21, 23, 24, 75, 76). It is not redundant to other clusters, and insertion of a single element into flamenco in D. melanogaster is sufficient to initiate silencing. Here we show that the function of flamenco appears to have diversified in the D. simulans clade, acting in at least some tissues as a germline piRNA cluster.

Dual stranded expression of flamenco

In this work, we showed that piRNAs of the flamenco locus in D.simulans and D. mauritiana are deposited maternally, align to both strands, and exhibit ping-pong signatures. This is in contrast to D. melanogaster, where flamenco acts as a uni-strand cluster in the soma (3), our data thus suggest that the flamenco locus in D. simulans and D. mauritiana acts as a dual-strand cluster in the germline. In D. sechellia the attributes of flamenco uncovered in D. melanogaster appear to be conserved – no expression in the maternal fraction and the testis and no ping pong signals. Given that flamenco is likely a somatic uni-strand cluster in D. erecta, we speculate that the conversion into a germline cluster happened in the simulans clade (3). Such a conversion of a cluster between the somatic and the germline piRNA pathway is not unprecedented. For example, a single insertion of a reporter transgene triggered the conversion of the uni-stranded cluster 20A in D. melanogaster into a dual-strand cluster (77).

The role of flamenco in D. simulans and D. mauritiana as the master regulator of piRNA in somatic support cells may still well be true – the promoter region of the flamenco cluster is conserved between species and between copies of flamenco within species. This suggests that in at least some contexts (or all) the cluster is still serving as a uni-strand cluster transcribed from a traditional RNA Pol II site (15). However it has acquired additional roles, producing dual strand piRNA and ping pong signals, in these two species, in at least the germline. However, in D. simulans, the majority of these reverse stranded piRNAs are emerging from the R1 insertions within flamenco. There is no evidence at present that R1 has undergone an expansion in function in D. simulans, thus it is unclear what, if any, functional impact the reverse stranded piRNAs have at the flamenco locus.

Duplication of flamenco in D. simulans

In D. simulans, flamenco is present in 2 genomic copies, and this duplication is present in all sequenced D. simulans lines except the reference strain. The dip1 gene and putative flamenco promoter flanking the duplication also has a high similarity in all sequenced lines (Fig. 2B). This raises the possibility that the duplication of flamenco in D. simulans was positively selected. Such a duplication may be beneficial as it increases the ability of an organism to rapidly silence TEs. Individuals with large piRNA clusters (or duplicated ones) will accumulate fewer deleterious TE insertions than individuals with small clusters (or non-duplicated ones), and duplicated clusters may therefore confer a selective advantage (78).

Rapid evolution of piRNA clusters

A previous work showed that dual- and uni-strand clusters evolve rapidly in Drosophila (20). In agreement with this work we also found that the flamenco-locus is rapidly evolving between and within species (Fig. 1C, 3B). A major open question remains whether this rapid turnover is driven by selection (positive or negative) or an outcome of neutral processes (eg. high TE activity or insertion bias of TEs). These rapid evolutionary changes at the flamenco locus, a piRNA master locus, suggest that there is a constant turnover in patterns of piRNA biogenesis that potentially leads to changes in the level of transposition control between individuals in a population.

Funding

This work was supported by the National Science Foundation Established Program to Stimulate Competitive Research (NSF-EPSCoR-1826834 and NSF-EPSCoR-2032756) to SS and the Austrian Science Fund FWF (https://www.fwf.ac.at/;) grant P35093 to RK. JV was supported by a Pathway to Independence award from the National Institute of General Medical Sciences (K99-GM137077). BYK was supported by the NIH-NRSA F32GM135998. ECL was supported by the National Institute of General Medical Sciences (R01-GM083300) and National Institutes of Health MSK Core Grant (P30-CA008748).

Competing interests

We declare that we have no competing interests.

Authors’ contributions

SS conceived the study, performed bioinformatics and drafted portions of the manuscript. FW and RK performed bioinformatics and drafted portions of the manuscript. JV contributed data and bioinformatic analysis. ECL drafted portions of the manuscript and provided data. BYK generated and contributed sequence data.

Availability of data and materials

All data has been made available in the following repositories:

Acknowledgements

Thanks to Colin Meiklejohn for providing some of the fly strains used in this manuscript. We would also like to thank Dimitri Petrov and his lab for providing logistical support to BYK. SS would like to thank Jeff Kittilson for assistance in the laboratory. SS would also like to thank C & F & S Emery for insightful commentary on the manuscript.

Footnotes

  • An author was added, Figures 1 and 5 were revised.

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Posted November 17, 2022.
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Rapid evolutionary diversification of the flamenco locus across simulans clade Drosophila species
Sarah Signor, Jeffrey Vedanayagam, Bernard Y. Kim, Filip Wierzbicki, Robert Kofler, Eric C. Lai
bioRxiv 2022.09.29.510127; doi: https://doi.org/10.1101/2022.09.29.510127
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