Abstract
Sexual reproduction evolved 1-2 billion years ago and underlies the biodiversity of our planet. Nevertheless, devolution of sexual into asexual reproduction can occur across all phyla of the animal kingdom. The genetic basis for how parthenogenesis can arise is completely unknown. To understand the mechanism and benefits of parthenogenesis, we have sequenced the genome of the facultative parthenogen, Drosophila mercatorum, and compared its organisation and expression pattern during parthenogenetic or sexual reproduction. We identified three genes, desat2, Myc, and polo in parthenogenetic D. mercatorum that when mis-regulated in a non-parthenogenetic species, D. melanogaster, enable facultative parthenogenetic reproduction. This simple genetic switch leads us to propose that sporadic facultative parthenogenesis could evolve as an ‘escape route’ preserving the genetic lineage in the face of sexual isolation.
Introduction
Parthenogenesis is a form of reproduction resulting in uniparental offspring having only the maternal genome; it is a virgin birth. There are two types of parthenogenesis: facultative, having the ability to switch back to sexual reproduction; and obligate, in which this is not possible. Sexual reproduction requires a carefully orchestrated program whereby the genome is first duplicated before undergoing two divisions in the absence of DNA synthesis to generate a complement of haploid gametes that can be combined with those of the opposite sex, or mating type in the context of lower eukaryotes, to generate a diploid zygote.
Facultative parthenogens retain the key meiotic machinery and yet have a hitherto unknown, but likely heritable, change that enables them to regain diploidy after meiosis and initiate mitotic divisions. By contrast, obligate parthenogens can theoretically have a block anywhere in meiosis and may eliminate it completely. It is therefore likely that different mechanisms underlie parthenogenesis depending upon which stage of sexual reproduction is blocked.
Parthenogenesis was first observed in aphids by Charles Bonnet in approximately 1740 and yet, its underlying mechanism has not been identified in any animal. Despite being poorly understood, parthenogenesis is generally regarded as being a deleterious reproductive strategy because it fails to generate genetic diversity. Nevertheless, parthenogenesis has evolved repeatedly across different phyla of animals and plants. One reason for the failure to identify any genetic cause of naturally occurring parthenogenesis in animals is that ancient obligate parthenogenetic lineages are often compared to similar, sexually reproducing counterparts that have sometimes diverged millions of years ago. It then becomes impossible to separate the primary cause from multiple downstream consequences. If we are to understand parthenogenesis, we must look at new species or, better yet, examine those able to switch from sexual to parthenogenetic reproduction. We postulated that a genetic cause likely underlies facultative parthenogenesis because it can undergo selection in Drosophila, locuts, and chickens and increase in frequency over several generations [1–4]. We therefore sought to uncover the genetic cause behind facultative parthenogenesis in Drosophila mercatorum, by sequencing its genome and comparing gene expression patterns during the oogenesis of females undertaking sexual or parthenogenetic reproduction. We, and now report a genetic cause of sporadic facultative parthenogenesis in D. mercatorum and show how these traits can be transferred to a sexually reproducing species, Drosophila melanogaster.
Results
The parthenogenetic ability of D. mercatorum
The facultative parthenogen, D. mercatorum, is unique in that some strains can behave as obligate parthenogens upon transitioning to parthenogenetic reproduction and can then be maintained in the lab indefinitely as healthy and easily expandable female only stocks [4–6].
D. mercatorum belongs to the repleta species group of South American cactus feeders which are approximately 47 My diverged from D. melanogaster [7]. However, D. mercatorum appears invasive and has spread, far beyond the range of most other repleta, to Australia and as far north as New York [4, 8]. As nearly all strains of D. mercatorum studied to date show some degree of parthenogenetic capability [4], we began by determining the baseline of parthenogenesis in 8 different D. mercatorum strains using a classical assay adapted from the first study of Drosophila parthenogenesis [3]. Large numbers of virgin females were maintained on fresh food for the duration of their lives and the food examined for offspring at any developmental stage. The numbers of progeny ranged from the generation of a small number of developing embryos that died before hatching to the production of a small number of fertile adult flies (Table S1). We observed that parthenogenetic offspring were produced from middle aged mothers (Table S1). We also confirmed by PCR with general Wolbachia primers that there was no Wolbachia infection (Table S1), since it is known to cause parthenogenesis in other arthropods [9], although Wolbachia is only known to cause cytoplasmic incompatibility in Drosophila [10]. We also confirmed that the strains examined were indeed all D. mercatorum since they were able to interbreed producing viable and fertile male and female offspring, although the parthenogenetically reproducing strain had slight impediment to breeding and did not consistently produce offspring (Table S2). As a result of these experiments, we selected two D. mercatorum stains for further study, a parthenogenetic strain from Hawaii and a sexually reproducing strain with very low parthenogenetic capability from São Paulo, Brazil.
The genome of D. mercatorum
In search for genetic changes permitting parthenogenesis, we chose to sequence and compare the genomes of the chosen sexually reproducing and parthenogenetic strains of D. mercatorum. We produced polished chromosome-level genome assemblies, using Oxford Nanopore Technology (ONT) and Illumina sequencing technology, that were then annotated (Fig. 1A). Most of the genes were on the 14 largest contigs (Fig. S1A-B). We ensured that the sequencing depth and coverage were uniform by plotting the reads over the assembled genome (Fig. S1C-D). The quality of the assemblies was assessed using standard metrics of N50, coverage, genome size, and gene content (Fig. 1A). When aligned, the sexual and parthenogenetic genomes were highly similar having only 1.2% divergence (Fig. 1B), which is consistent with pairwise heterozygosity, and thus further confirming that they are indeed the same species. We observed inversions on the 2L chromosome arm which had previously been noticed between D. mercatorum populations collected from South and North America [8, 11].
A) Genome assembly data metrics, quality control, and annotation metrics B) Alignment of the parthenogenetic D. mercatorum genome against the sexual genome.
We found 24.4% divergence between both parthenogenetically and sexually reproducing D. meractorum genome assemblies and the D. melanogaster reference genome (release 6).
There was clustering of each contig from both D. meractorum genomes to specific chromosome arms in D. melanogaster (Fig. S2A-B), indicative of the shuffling of genes, which largely remain on the same chromosome arms. We also confirmed chromosome arm contig matching by checking the DNA k-mers using Nucmer (Fig. S3A-B). This accords with long-held knowledge of how corresponding chromosome arms form a series of homologous genetic ‘building blocks’ in different Drosophila species within which synteny is lost [12, 13]. These chromosome arm ‘building blocks’ correspond to the six Muller elements (A-F) and are conserved across Diptera [14]. Together these analyses indicated that the chromosome-level genome assemblies for the sexually and parthenogenetically reproducing D. mercatorum strains were suited to detailed comparison between each other and with the D. melanogaster genome.
We next confirmed that the genome assemblies matched the karyotypes of the sexual and parthenogenetic of D. mercatorum strains by localising local sequence markers onto preparations of mitotic chromosomes from D. mercatorum third instar larval brains using a Hybridisation Chain reaction (HCR) fluorescence in situ hybridization (FISH) protocol that we developed for this purpose (see Supplementary text). We selected single genes within syntenic blocks that are conserved between D. melanogaster and D. mercatorum to serve as markers for each of the 6 chromosome arms of the mitotic karyotype (Fig. 2A). This allowed us to identify the Muller elements, A-F, for both sexual and parthenogenetic D. mercatorum strains (Fig. 2B-C). We found a fusion of the 2L/B and 3R/E (D. melanogaster/Muller) chromosome arms that was previously documented as unique to D. mercatorum within the repleta group [11] and the remaining chromosome arms were telocentric. We observed that the 4th chromosome of the parthenogenetic strain was substantially larger than the 4th chromosome in the sexual strain and we continue to investigate the underlying reason for this. We also used HCR FISH to physically position the 14 largest contigs from the parthenogenetic genome onto the 3rd instar larval salivary gland polytene chromosomes of both the sexual and parthenogenetic strains of D. mercatorum. We found that each contig mapped to the chromosome arm as predicted by the annotation and nucleotide sequence (Fig. 2D-F). Notably the polytene 4th (F) chromosome of sexually and parthenogenetically reproducing strains of D. mercatorum appeared of similar size suggesting that the increased size of their corresponding diploid chromosomes is due to acquisition of satellite, heterochromatic sequences that do not undergo endoreduplication in the generation of polytene chromosomes. We conclude that the two chromosome-level genome assemblies of the sexually and parthenogenetically reproducing D. mercatorum represent the protein coding part of these genomes and accurately reflects chromosome organisation in this species.
A) Select genes used for probes to identify the chromosome arms. B) Mitotic chromosomes with the chromosome arm indicated for the parthenogenetic and sexually reproducing D. mercatorum 3rd instar neuroblasts stained with DAPI and the probes indicated in ‘A’, chromosomes are outlined with a white dashed line. The indicated chromosome was marked in 100% of typical karyotypes analysed (n≥42, N≥3). The scale is 1μm. C) Cartoon of the parthenogenetic and sexual D. mercatorum karyotype with the analogous D. melanogaster chromosomes indicated along with the Muller Element letter in brackets. D) Select genes that have chromosome level synteny across the genus Drosophila that were used to make probes for mapping contigs to the polytene chromosomes. E) Images of polytene chromosomes used for mapping of the largest 14 contigs of the sexual and parthenogenetic D. mercatorum genomes, chromosomes were stained with DAPI and HCR in situ DNA probes matching the genes listed in ‘B’. The scale is 10μm. F) Schematic of the mapping of the first 14 Contigs.
Gene expression differences between sexual and parthenogenetic D. mercatorum
We argued that genomic changes with potential to lead to changes in reproductive ability should reveal themselves as gene expression changes late in female germline development. We therefore used RNA sequencing to characterise the transcriptomes of mature eggs (Stage 14 egg chambers) isolated from the sexual, parthenogenetic, and a ‘partially parthenogenetic’ strain of D. mercatorum. The partially parthenogenetic strain reproduces sexually but has an enhanced ability to switch to parthenogenetic reproduction. From the three transcription profiles we identified 7656 genes that were expressed in mature eggs with the same distribution as the annotated genes (Fig. S4A-B, Fig. S1A-B). There were 92 genes differentially expressed in all three pairwise transcriptome comparisons (Fig. S4C, Tables S3). However, there were few strongly and significantly differentially expressed genes and after manual curation a subset were selected for further study, highlighted in the volcano plots (Fig. S4D). We analysed the significantly differentially expressed genes from all pairwise comparisons and found gene ontology (GO) enrichment of genes involved in redox, immune function, wing disc growth, biosynthesis, proteolysis, and translation (Fig. S5A). Following consideration of the GO analysis and the pairwise comparisons of expression, we selected a further set of genes whose mis-regulation could potentially result in parthenogenesis (Fig. 3A). Genes were selected that exhibited significant differential expression (padj<0.05) at a level equivalent to heterozygosis (log2 fold change ± 0.6) or greater and were involved in common cellular processes, based upon the gene ontology analysis.
A) Differentially expressed genes between the parthenogenetic, partially parthenogenetic, and sexual transcriptomes, the function was assigned from flybase.org, the log2 fold change and the padj are given for only the parthenogenetic vs sexual comparison except those with a (*) are from the partial parthenogenetic vs sexual comparison, the screening was performed with the listed genetic tools. B) Biased screen of cell cycle and centrosome genes that were not differentially expresses, the function was attributed from flybase.org and the screening was performed with the listed genetic tools. D) Negative controls that were not differentially expressed, the screening was performed with the listed genetic tools.
Functional screens for parthenogenesis
We decided to take a two-pronged approach in an effort to identify genes that could lead to parthenogenesis in a sexually reproducing fly: an unbiased screen of candidate genes from the transcriptomics (Fig. 3A), a biased screen of candidate genes based on their cell cycle/centriole functions (Fig. 3B), and a series of controls (Fig. 3C). For the first group, our objective was to replicate, as far as possible, the degree of differential expression seen between D. meractorum strains (Fig. S6). Since all strains of D. mercatorum we screened were already parthenogenetic to some degree, we carried out this screen in the non-parthenogenetic species D. melanogaster. Using 13 different Drosophila species, we first determined that a baseline indicator of parthenogenesis could be given by testing the ability of approximately 500 virgin female flies to generate progeny (Table S4). Strong levels of parthenogenesis could be detected with as little as 30 flies. Using these criteria, we found that two typical laboratory strains of D. melanogaster (w− and Oregon-R) showed no parthenogenesis whatsoever, whereas a strain caught in the wild (CB1) produced a small number of embryos that showed restricted development before dying (Table S5). This accords with previous findings that D. melanogaster strains caught in the wild have slight parthenogenetic ability [3].
We then tested whether down-regulating the D. melanogaster homologs of genes showing reduced expression in parthenogenetic D. mercatorum strains would result in the production of offspring that died as embryos, larvae, pupae, or from old age as adult flies. To this end, we examined CRISPR knock-out alleles that we generated in candidate genes (Fig. S7, Table S6); publicly available mutants; or established lines in which candidate genes were down-regulated by RNAi. We also tested D. melanogaster constructs engineered to increase expression of genes whose homologues had elevated expression in the parthenogenetic D. mercatorum strains. In the case of variant alleles that were not homozygous viable, screening was carried out on heterozygotes. Together we screened a total of 44 genes (Fig. 3A-C, see Supplementary text) and identified 16 able to cause 0.1-0.4% parthenogenesis in D. melanogaster when their expression was either increased or decreased (Fig. S8A; Table S7 and see Supplementary text). The parthenogenesis observed resulted in the offspring developing to varying stages and dying as embryos or from old age as adult flies. The percentage given is relative to the number of adult flies screened. For this single mutant screen, we found largely only the generation of embryos that died before hatching.
The low level of parthenogenesis detected in this single mutant screen, where the expression of only a single gene was perturbed, is in line with earlier studies that had concluded that parthenogenesis was a polygenic trait [4]. This consideration led us to carry out a double mutant screen in which we combined pairs of variants in different genes into individual fly stocks that we then screened for parthenogenesis. This revealed several mutant combinations able to generate between 0.5-7% parthenogenetic offspring that died as embryos, larvae, pupae, or from old age as adult flies. From the more successful combinations, we found that one of the mutant genes either encoded a desaturase, desat1 or desat2, or a protein predominantly involved in regulating cell division and proliferation, Myc, slmb, or polo (Fig. 4A, and Table S8). Notably, 0.8% of the offspring derived from females heterozygous for a mutation in desat2 and carrying two extra copies of a polo transgene expressed from its endogenous promotor (GFP-polo4+;; desat1−/+) developed to adulthood (Table S8). This level of D. melanogaster parthenogenesis is a comparable the ‘partially parthenogenetic’ strain of D. mercatorum used in generating the transcriptomics data. Parthenogenesis results from decreased expression of either desat1 or desat2. Since the desat2 allele is known to be a natural variant present in most populations, we determined whether our desat1 stock carries the desat2 allele and indeed it does. Therefore, the desat1 stock is a double mutant for desat1 and desat2, accounting for its stronger phenotype.
A) Positive results from the functional screens of candidate genes that may cause parthenogenesis and two controls, the p-value was calculated using the Fisher’s exact test the control used for these calculations was the combined results from the primary screen for the two genes used.
We then asked whether the parthenogenetic offspring obtained from these screens for parthenogenesis in D. melanogaster were themselves able to carry out parthenogenetic reproduction and found that none of them could (Tables S7-8). We did, however, find that the parthenogenetic D. melanogaster were still able to mate with males and produce fertile offspring (Fig. S8B), similar to previous findings [4]. The parthenogenetic D. mercatorum offspring from the sexually reproducing stocks could not be established as a lab stock and did not survive beyond the 7th generation of parthenogenesis as also found previously [15]. Even our long-held stocks of fully parthenogenetic females were able to mate with males (Table S2). Therefore, we have not found a genetic combination that leads to obligate-like parthenogenesis, but we have identified key genes for facultative parthenogenesis.
Having identified D. mercatorum genes whose homologues led to a degree of parthenogenetic development when mis-expressed in D. melanogaster, we looked for genomic differences in these genes in sexual and parthenogenetic strains of D. mercatorum. We found no substantial changes in gene organisation of desat1/2, polo, or slmb, although we cannot exclude the possibility of changes in distal enhancer elements that have not been mapped (Fig. S9A-D, Supplementary text). There were several changes to the Myc locus that could affect the expression of the protein and change its downstream function (Fig. S9E). The Myc locus of the parthenogenetic strain showed many deletions and insertions leading to the changes in primary amino-acid sequence of the protein as indicated in Fig. S10A. None of these mutations affected either the basic Helix-Loop-Helix (bHLH) DNA-binding domain or the three Myc Box (MB) domains (1-3), or the three known phosphorylation sites of the Myc protein (Fig. S9E) [16–18]. There were also changes in genome organisation at the Myc locus of the D. mercatorum parthenogenetic genome, which has a 1.4kbp repetitive region between a Drosophila INterspersed Elements-1 (DINE-1/INE1) transposable element (TE) and the Myc coding region, which are 9.3kbp apart. A similar TE insertion in the classic mutant allele of Myc (dm1) causes the minute phenotype [16], therefore having a repressive effect on Myc expression. The 1.4kbp repetitive sequence present in the parthenogenetic genome between the gene and the TE could allow de-repression resulting in an increased expression of Myc relative to the sexually reproducing flies. Finally, we detected a 48bp deletion in the parthenogenetic genome 344bp up stream of the start site. The above mutations have the potential to affect the transcription, translations, or protein stability of Myc. Moreover, they might also perturb Myc’s functions as a transcription factor to influence the expression of the other genes identified in our study [19]. Future studies will be required to distinguish these possibilities.
The development of parthenogenetic embryos
To understand how development might be initiated during parthenogenesis we first examined fertilised eggs from the sexually reproducing D. mercatorum strain that were initiating the mitotic nuclear division cycles (Fig 5C, Fig. S11A) to compare to the parthenogenetic eggs. The extent to which nuclear division cycles could take place in parthenogenetic D. mercatorum reflected the extent of parthenogenicity. We observed that 38% of unfertilised eggs from the parthenogenetic strain had one or more cell divisions (Fig 5A,C, Fig. S11E-G), whereas 12% of unfertilised eggs from the partially parthenogenetic strain showed one or more cell divisions (Fig 5B,C, Fig. S11B-D). The sexually reproducing D. mercatorum embryos had timely cell divisions and no obvious nuclear defects. In contrast, the unfertilised parthenogenetic and partially parthenogenetic embryos developed with abnormal numbers of nuclei (Fig. S11D) or DNA abnormalities (Fig. S11G). All parthenogenetic Drosophilids appear to retain normal meiosis and rediploidise their genomes either by fusion of one or more of the four haploid nuclei arising from meiosis or by post-meiotic duplication of the haploid gamete [3, 5, 20–24]. All four meiotic products, three polar bodies and the female pronucleus, are present within the Drosophilid egg, and the three polar normally fuse and arrest in a mitotic-like state. We only observed the presence of polar bodies in 44% of parthenogenetic embryos that initiated the mitotic nuclear division cycles suggesting that the missing polar bodies may be participating in the mitotic nuclear division cycles in the developing embryos.
A-B) Unfertilised parthenogenetic and partially parthenogenetic D. mercatorum embryos. The parthenogenetic embryo has entered the mitotic divisions. The partially parthenogenetic embryo has initiated the first mitosis. Refer to Fig. S11. C) Histogram of the proportion of sexually and parthenogenetically reproducing D mercatorum eggs/embryos that have aggregated polar bodies, initiated the first mitosis, or have entered the mitotic cell divisions. E-D) GFP-polo4+, desat2−/−, and GFP-polo4+; desat2−/+ D. melanogaster embryos that have initiated the first mitosis, aggregated polar bodies, and have entered the mitotic cell divisions, respectively. G-M) GFP-polo4+, desat2−/−, and GFP-polo4+; desat2−/+ D. melanogaster embryos that have initiated the first mitosis, aggregated polar bodies, and have entered the mitotic cell divisions. N) Histogram of the proportion of wildtype unfertilised and fertilised D. melanogaster eggs/embryos that have aggregated polar bodies, initiated the first mitosis, or have entered the mitotic cell divisions, refer to Fig. S12 for examples. O) Histogram of the proportion of unfertilised GFP-polo4+, desat2−/−, and GFP-polo4+; desat2−/+ D. melanogaster embryos that have aggregated polar bodies, initiated the first mitosis, or have entered the mitotic cell divisions. The Fisher’s exact test was used to calculate all p-values. The nuclei are marked with asterisks. The scale is 10μm.
We then examined fertilised and unfertilised wildtype D. melanogaster eggs and compared their development to the parthenogenetic D. melanogaster eggs. All unfertilised wildtype D. melanogaster eggs completed meiosis and 70% appeared to have entered the first mitotic division and had condensed chromosomes (Fig 5N, Fig. S12A-B). In contrast, nearly all fertilised embryos had begun to undergo the mitotic nuclear division cycles (Fig 5N, Fig. S11C-D). Development of the induced parthenogenetic D. melanogaster embryos mirrored the findings from the parthenogenesis screens. We found nearly all unfertilised eggs laid by either GFP-polo4+ or desat2−/− mothers were not able to undertake mitotic nuclear division cycles (Fig. 5D-E,G-J,O, Fig. S13A-D). By contrast, 6% of the unfertilised eggs laid by GFP-polo4+;; desat2−/+ D. melanogaster mothers could undertake at least limited mitosis (Fig. 5F,K-M,O, Fig. S13E-G). Similar to the unfertilised parthenogenetic and partially parthenogenetic D. mercatorum embryos, the parthenogenetic D. melanogaster embryos had abnormal numbers of nuclei and/or DNA abnormalities. Although there are abnormalities in these parthenogenetic embryos, during early embryogenesis there only need be one normal nucleus dividing to produce an animal. We were unable to observe polar bodies in the unfertilised GFP-polo4+;; desat2−/+ - derived eggs that had initiated the mitotic cell divisions and found that nuclei were not present in the expected ratio, suggesting that the polar bodies participate in the nuclear division cycles. This leads us to propose that the recapture of polar bodies contributes to rediploidisation in these induced parthenogenetic D. melanogaster embryos.
Discussion
Our study offers the first account of a molecular basis underlying the evolution of any type of parthenogenesis in any animal. Our findings relate specifically to the Dipteran D. mercatorum and suggest a route through which parthenogenesis could arise in this species.
Key to this is the differential expression of desat and cell cycle genes between parthenogenetically and sexually reproducing strains. The ability of desat1 mutants to enhance the phenotype of desat2 mutants in driving parthenogenesis when heterozygous in D. melanogaster is likely a consequence of overlapping function between their encoded proteins, which show 85% identity in amino-acid sequence. Both desat1 and desat2 encode desaturases that generate double bonds in hydrocarbons and have roles in lipid metabolism. Desat1 also generates double bonds during sex pheromone biogenesis [25], and desat1 mutations can result in female resistance to mating. Desat2 desaturates cuticular hydrocarbons; it has been associated with increased cold tolerance [26] and is credited with imparting the ability of D. melanogaster to become invasive and colonise cosmopolitan habitats. The highly pleiotropic nature of desat1 and desat2 makes it difficult to determine how their down-regulation relates to the increased incidence of parthenogenesis. As their mutation leads to a higher fluidity of membranes, it is possible that this could influence a wide range of membrane associated trafficking events associated with the completion of female meiosis, behaviour of the polar bodies, and the onset of zygotic mitoses. The potential effects of these mutations upon such events will require detailed future studies.
The other group of genes involved in enabling parthenogenesis regulate some aspect of cell cycle progression. Slmb is a subunit of the SCF, the Skp, Cullin, F-box containing ubiquitin ligase complex that regulates S phase entry by targeting multiple substrates. One of the multiple targets of the SCF is Myc [27]. There was only a modest increase in Myc transcripts (0.6 Log2 fold change) in the parthenogenetic strain, however, this expression level change was highly significant (padj<0.001) and is equivalent to having one extra copy of the gene.
The Myc bHLH transcription factor has been shown to give D. melanogaster cells a competitive growth advantage [28] and could account for the finding that parthenogenetic offspring are physically larger than the sexually reproducing animals [29]. Myc is known to promote Polo-like-kinase1 expression in mammalian cells that in turn destabilises SCF [30]. If a similar mechanism were to act in Drosophila, this could account for the relationships we observe here to promote parthenogenesis. Our findings suggest that an effective step towards establishing parthenogenesis is the heterozygosity of desat1 or desat2 coupled to Myc over-expression or heterozygosity of the SCF (slmb).
Although we have shown that we can induce parthenogenesis in a sexually reproducing line of D. melanogaster to a similar degree as a partially parthenogenetic strain of D. mercatorum, we are not able to maintain these animals as a parthenogenetic stock. Therefore, although we identify a significant step towards heritable parthenogenesis, this is not the end of the story and additional changes would be required for parthenogenesis to become fixed in a population and transit to more obligate-like parthenogenetic reproduction. Moreover, there are likely to be many alternative paths to the devolution of sexual reproduction in animals and this could explain the varying degree of parthenogenetic ability, not only within D. mercatorum, but also across the Drosophila genus. Given the polygenic nature of facultative parthenogenesis and the fact that there are multiple inputs into core cell cycle regulation, it may explain why no unifying signature of parthenogenesis has been found to date [31]. Thus, we anticipate that parthenogenesis might have different causal events in each species or even between individuals of the same species.
Some consider sporadic facultative parthenogenesis to be an unimportant accident. However, there could be a benefit of having sporadic facultative parthenogenesis inducing heterozygotic mutations floating around in the population, they may facilitate an ‘extinction escape hatch’, thus helping a lineage of the species stave off extinction in the face of isolation until an opportunity to mate arises again. Parthenogenesis is spread across the order Diptera and rare facultative parthenogenesis is prevalent in Drosophila [32] making it likely that the mechanism we propose is not restricted to D. mercatorum.
Acknowledgements
We would like to thank Jonathan Day (Frank Jiggens’ Lab) and Bettina Fischer (Richard Durbin’s Lab) for advice on library preparation, technical assistance, protocols, reagents, and endless patience. We would like to thank Harry Choi and Mike Liu, from Molecular Instruments for troubleshooting and for gifted reagents that were during the optimisation of the HCR FISH protocol for use on DNA. We would like to thank Paula Almeida-Coelho for technical advice on in situs and karyotyping. We would like to thank Richard Durbin for advice, discussions, and computation assistance. We would like to thank Frank Sprenger for flies, kindly brought to Cambridge. Finally, we would like to thank the Genetics Fly Facility for embryo injections, particularly Dr Alla Madich for multiple attempts at developing transgenics in D. mercatorum.
Funding
We gratefully acknowledge support from Leverhulme Trust Project Grant RPG-2018-229, (ALB, DKF and DMG). We gratefully acknowledge support from NIH/NIDA U01DA047638 (EG) and NIH/NIGMS R01GM123489 (EG)
Author contributions
Conceptualization: ALB
Methodology: ALB, DKF, EG, DMG
Investigation: ALB, DKF, EG
Visualization: ALB, DKF, EG
Funding acquisition: ALB, DMG
Project administration: ALB, DMG
Supervision: DMG
Writing – original draft: ALB
Writing – review & editing: DKF, EG, DMG
Competing interests
Authors declare that they have no competing interests.
Data and materials availability
All raw and analyzed D. mercatorum genomic and transcriptomic data generated by this study will be provided on public databases, all code used for analysis in on GitHub via FabianDK and ekg, all Drosophila strains/stocks will be offered to Bloomington Stock Centre or made available upon request.
