Phased Assembly of Neo-Sex Chromosomes Reveals Extensive Y Degeneration and Rapid Genome Evolution in Rumex hastatulus.

Y chromosomes are thought to undergo progressive degeneration due to stepwise loss of recombination and subsequent reduction in selection efficiency. However, the timescales and evolutionary forces driving degeneration remain unclear. To investigate the evolution of sex chromosomes on multiple timescales, we generated a high-quality phased genome assembly of the massive older (<10 MYA) and neo (<200,000 yr) sex chromosomes in the XYY cytotype of the dioecious plant Rumex hastatulus and a hermaphroditic outgroup Rumex salicifolius. Our assemblies, supported by fluorescence in situ hybridization, confirmed that the neo-sex chromosomes were formed by two key events: an X-autosome fusion and a reciprocal translocation between the homologous autosome and the Y chromosome. The enormous sex-linked regions of the X (296 Mb) and two Y chromosomes (503 Mb) both evolved from large repeat-rich genomic regions with low recombination; however, the complete loss of recombination on the Y still led to over 30% gene loss and major rearrangements. In the older sex-linked region, there has been a significant increase in transposable element abundance, even into and near genes. In the neo-sex-linked regions, we observed evidence of extensive rearrangements without gene degeneration and loss. Overall, we inferred significant degeneration during the first 10 million years of Y chromosome evolution but not on very short timescales. Our results indicate that even when sex chromosomes emerge from repetitive regions of already-low recombination, the complete loss of recombination on the Y chromosome still leads to a substantial increase in repetitive element content and gene degeneration.

Non-canonical bases differentially represented in the sex chromosomes of the dioecious plant Silene latifolia.

The oxidation of 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC), and 5-carboxylcytosine (5caC), known as oxi-mCs, garners significant interest in plants as potential epigenetic marks. While research in mammals has established a role in cell reprogramming, carcinogenesis, and gene regulation, their functions in plants remain unclear. In rice, 5hmC has been associated with transposable elements (TEs) and heterochromatin. This study utilizes Silene latifolia, a dioecious plant with heteromorphic sex chromosomes and a genome with a large proportion of TEs, which provides a favourable environment for the study of oxi-mCs in individual sexes. Notably, we detected surprisingly high levels of oxi-mCs in S. latifolia comparable with mammals. Nuclei showed enrichment in heterochromatic regions, except for 5hmC whose signal was homogeneously distributed. Intriguingly, the same X chromosome in females displayed overall enrichment of 5hmC and 5fC compared with its counterpart. This fact is shared with 5mC, resembling dosage compensation. Co-localization showed higher correlation between 5mC and 5fC than with 5hmC, indicating no potential relationship between 5hmC and 5fC. Additionally, the promoter of several sex-linked genes and sex-biased TEs clustered in a clear sex-dependent way. Together, these findings unveil a hypothetical role for oxi-mCs in S. latifolia sex chromosome development, warranting further exploration.

Sexy ways: the methodical approaches to study plant sex chromosomes.

Sex chromosomes have evolved in many plant species with separate sexes. Current plant research is shifting from examining the structure of sex chromosomes to exploring their functional aspects. New studies are progressively unveiling the specific genetic and epigenetic mechanisms responsible for shaping distinct sexes in plants. While the fundamental methods of molecular biology and genomics are generally employed for the analysis of sex chromosomes, it is often necessary to modify classical procedures not only to simplify and expedite analyses but sometimes to make them possible at all. In this review, we demonstrate how, at the level of structural and functional genetics, cytogenetics, and bioinformatics, it is essential to adapt established procedures for sex chromosome analysis.

HiC-TE: a computational pipeline for Hi-C data analysis to study the role of repeat family interactions in the genome 3D organization.

MOTIVATION: The role of repetitive DNA in the 3D organization of the interphase nucleus is a subject of intensive study. In studies of 3D nucleus organization, mutual contacts of various loci can be identified by Hi-C sequencing. Typical analyses use binning of read pairs by location to reduce noise. We use binning by repeat families instead to make similar conclusions about repeat regions. RESULTS: To achieve this, we combined Hi-C data, reference genome data and tools for repeat analysis into a Nextflow pipeline identifying and quantifying the contacts of specific repeat families. As an output, our pipeline produces heatmaps showing contact frequency and circular diagrams visualizing repeat contact localization. Using our pipeline with tomato data, we revealed the preferential homotypic interactions of ribosomal DNA, centromeric satellites and some LTR retrotransposon families and, as expected, little contact between organellar and nuclear DNA elements. While the pipeline can be applied to any eukaryotic genome, results in plants provide better coverage, since the built-in TE-greedy-nester software only detects tandems and LTR retrotransposons. Other repeats can be fed via GFF3 files. This pipeline represents a novel and reproducible way to analyze the role of repetitive elements in the 3D organization of genomes. AVAILABILITY AND IMPLEMENTATION: https://gitlab.fi.muni.cz/lexa/hic-te/. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Chemical genetics in Silene latifolia elucidate regulatory pathways involved in gynoecium development.

Dioecious plants possess diverse sex determination systems and unique mechanisms of reproductive organ development; however, little is known about how sex-linked genes shape the expression of regulatory cascades that lead to developmental differences between sexes. In Silene latifolia, a dioecious plant with stable dimorphism in floral traits, early experiments suggested that female-regulator genes act on the factors that determine the boundaries of the flower whorls. To identify these regulators, we sequenced the transcriptome of male flowers with fully developed gynoecia, induced by rapid demethylation in the parental generation. Eight candidates were found to have a positive role in gynoecium promotion, floral organ size, and whorl boundary, and affect the expression of class B MADS-box flower genes. To complement our transcriptome analysis, we closely examined the floral organs in their native state using field emission environmental scanning electron microscopy, and examined the differences between females and androhermaphrodites in their placenta and ovule organization. Our results reveal the regulatory pathways potentially involved in sex-specific flower development in the classical model of dioecy, S. latifolia. These pathways include previously hypothesized and unknown female-regulator genes that act on the factors that determine the flower boundaries, and a negative regulator of anther development, SUPERMAN-like (SlSUP).

The Sister Chromatid Division of the Heteromorphic Sex Chromosomes in Silene Species and Their Transmissibility towards the Mitosis.

Young sex chromosomes possess unique and ongoing dynamics that allow us to understand processes that have an impact on their evolution and divergence. The genus Silene includes species with evolutionarily young sex chromosomes, and two species of section Melandrium, namely Silene latifolia (24, XY) and Silene dioica (24, XY), are well-established models of sex chromosome evolution, Y chromosome degeneration, and sex determination. In both species, the X and Y chromosomes are strongly heteromorphic and differ in the genomic composition compared to the autosomes. It is generally accepted that for proper cell division, the longest chromosomal arm must not exceed half of the average length of the spindle axis at telophase. Yet, it is not clear what are the dynamics between males and females during mitosis and how the cell compensates for the presence of the large Y chromosome in one sex. Using hydroxyurea cell synchronization and 2D/3D microscopy, we determined the position of the sex chromosomes during the mitotic cell cycle and determined the upper limit for the expansion of sex chromosome non-recombining region. Using 3D specimen preparations, we found that the velocity of the large chromosomes is compensated by the distant positioning from the central interpolar axis, confirming previous mathematical modulations.

Fundamentally different repetitive element composition of sex chromosomes in Rumex acetosa.

BACKGROUND AND AIMS: Dioecious species with well-established sex chromosomes are rare in the plant kingdom. Most sex chromosomes increase in size but no comprehensive analysis of the kind of sequences that drive this expansion has been presented. Here we analyse sex chromosome structure in common sorrel (Rumex acetosa), a dioecious plant with XY1Y2 sex determination, and we provide the first chromosome-specific repeatome analysis for a plant species possessing sex chromosomes. METHODS: We flow-sorted and separately sequenced sex chromosomes and autosomes in R. acetosa using the two-dimensional fluorescence in situ hybridization in suspension (FISHIS) method and Illumina sequencing. We identified and quantified individual repeats using RepeatExplorer, Tandem Repeat Finder and the Tandem Repeats Analysis Program. We employed fluorescence in situ hybridization (FISH) to analyse the chromosomal localization of satellites and transposons. KEY RESULTS: We identified a number of novel satellites, which have, in a fashion similar to previously known satellites, significantly expanded on the Y chromosome but not as much on the X or on autosomes. Additionally, the size increase of Y chromosomes is caused by non-long terminal repeat (LTR) and LTR retrotransposons, while only the latter contribute to the enlargement of the X chromosome. However, the X chromosome is populated by different LTR retrotransposon lineages than those on Y chromosomes. CONCLUSIONS: The X and Y chromosomes have significantly diverged in terms of repeat composition. The lack of recombination probably contributed to the expansion of diverse satellites and microsatellites and faster fixation of newly inserted transposable elements (TEs) on the Y chromosomes. In addition, the X and Y chromosomes, despite similar total counts of TEs, differ significantly in the representation of individual TE lineages, which indicates that transposons proliferate preferentially in either the paternal or the maternal lineage.

Comparison of various approaches to detect algal culture contamination: a case study of Chlorella sp. contamination in a Phaeodactylum tricornutum culture.

Microalgal contamination in algal culture is a serious problem hampering the cultivation process, which can result in considerable economic and time losses. With the field of microalgal biotechnology on the rise, development of new tools for monitoring the cultures is of high importance. Here we present a case study of the detection of fast-growing green algae Chlorella vulgaris (as contaminant) in a diatom Phaeodactylum tricornutum culture using various approaches. We prepared mixed cultures of C. vulgaris and P. tricornutum in different cell-to-cell ratios in the range from 1:103 to 1:107. We compared the sensitivity among microscopy, cultivation-based technique, PCR, and qPCR. The detection of C. vulgaris contamination using light microscopy failed in samples containing cell ratios <1:105. Our results confirmed PCR/qPCR to provide the most reliable and sensitive results, with detection sensitivity close to 75 cells/mL. The method was similarly sensitive in a pure C. vulgaris culture as well as in a mixed culture containing 107-times more P. tricornutum cells. A next-generation sequencing analysis revealed a positive discrimination of C. vulgaris during DNA extraction. The method of cultivation media exchange from sea water to fresh water, preferred by the Chlorella contaminant, demonstrated a presence of the contaminant with a sensitivity comparable to PCR approaches, albeit with a much longer detection time. The results suggest that a qPCR/PCR-based approach is the best choice for an early warning in the commercial culturing of microalgae. This method can be conveniently complemented with the substitution-cultivation method to test the proliferating potential of the contaminant. KEY POINTS: • PCR-based protocol developed for detection of Chlorella cells. • Synergy of various approaches shows deeper insight into a presence of contaminants. • Positive/negative discrimination occurs during DNA extraction in mixed cultures. • Newly developed assays ready to use as in diagnostics of contamination.

Multiple horizontal transfers of nuclear ribosomal genes between phylogenetically distinct grass lineages.

The movement of nuclear DNA from one vascular plant species to another in the absence of fertilization is thought to be rare. Here, nonnative rRNA gene [ribosomal DNA (rDNA)] copies were identified in a set of 16 diploid barley (Hordeum) species; their origin was traceable via their internal transcribed spacer (ITS) sequence to five distinct Panicoideae genera, a lineage that split from the Pooideae about 60 Mya. Phylogenetic, cytogenetic, and genomic analyses implied that the nonnative sequences were acquired between 1 and 5 Mya after a series of multiple events, with the result that some current Hordeum sp. individuals harbor up to five different panicoid rDNA units in addition to the native Hordeum rDNA copies. There was no evidence that any of the nonnative rDNA units were transcribed; some showed indications of having been silenced via pseudogenization. A single copy of a Panicum sp. rDNA unit present in H. bogdanii had been interrupted by a native transposable element and was surrounded by about 70 kbp of mostly noncoding sequence of panicoid origin. The data suggest that horizontal gene transfer between vascular plants is not a rare event, that it is not necessarily restricted to one or a few genes only, and that it can be selectively neutral.

The distribution of epigenetic histone marks differs between the X and Y chromosomes in Silene latifolia.

MAIN CONCLUSION: Contrasting patterns of histone modifications between the X and Y chromosome in Silene latifolia show euchromatic histone mark depletion on the Y chromosome and indicate hyperactivation of one X chromosome in females. Silene latifolia (white campion) is a dioecious plant with heteromorphic sex chromosomes (24, XX in females and 24, XY in males), and a genetically degenerated Y chromosome that is 1.4 times larger than the X chromosome. Although the two sex chromosomes differ in their DNA content, information about epigenetic histone marks and evidence of their function are scarce. We performed immunolabeling experiments using antibodies specific for active and suppressive histone modifications as well as pericentromere-specific histone modifications. We show that the Y chromosome is partially depleted of histone modifications important for transcriptionally active chromatin, and carries these marks only in the pseudo-autosomal region, but that it is not enriched for suppressive and pericentromere histone marks. We also show that two of the active marks are specifically enriched in one of the X chromosomes in females and in the X chromosome in males. Our data support recent findings that genetic imprinting mediates dosage compensation of sex chromosomes in S. latifolia.

DNA methylation and genetic degeneration of the Y chromosome in the dioecious plant Silene latifolia.

BACKGROUND: S. latifolia is a model organism for the study of sex chromosome evolution in plants. Its sex chromosomes include large regions in which recombination became gradually suppressed. The regions tend to expand over time resulting in the formation of evolutionary strata. Non-recombination and later accumulation of repetitive sequences is a putative cause of the size increase in the Y chromosome. Gene decay and accumulation of repetitive DNA are identified as key evolutionary events. Transposons in the X and Y chromosomes are distributed differently and there is a regulation of transposon insertion by DNA methylation of the target sequences, this points to an important role of DNA methylation during sex chromosome evolution in Silene latifolia. The aim of this study was to elucidate whether the reduced expression of the Y allele in S. latifolia is caused by genetic degeneration or if the cause is methylation triggered by transposons and repetitive sequences. RESULTS: Gene expression analysis in S. latifolia males has shown expression bias in both X and Y alleles. To determine whether these differences are caused by genetic degeneration or methylation spread by transposons and repetitive sequences, we selected several sex-linked genes with varying degrees of degeneration and from different evolutionary strata. Immunoprecipitation of methylated DNA (MeDIP) from promoter, exon and intron regions was used and validated through bisulfite sequencing. We found DNA methylation in males, and only in the promoter of genes of stratum I (older). The Y alleles in genes of stratum I were methylation enriched compared to X alleles. There was also abundant and high percentage methylation in the CHH context in most sequences, indicating de novo methylation through the RdDM pathway. CONCLUSIONS: We speculate that TE accumulation and not gene decay is the cause of DNA methylation in the S. latifolia Y sex chromosome with influence on the process of heterochromatinization.

The slowdown of Y chromosome expansion in dioecious Silene latifolia due to DNA loss and male-specific silencing of retrotransposons.

BACKGROUND: The rise and fall of the Y chromosome was demonstrated in animals but plants often possess the large evolutionarily young Y chromosome that is thought has expanded recently. Break-even points dividing expansion and shrinkage phase of plant Y chromosome evolution are still to be determined. To assess the size dynamics of the Y chromosome, we studied intraspecific genome size variation and genome composition of male and female individuals in a dioecious plant Silene latifolia, a well-established model for sex-chromosomes evolution. RESULTS: Our genome size data are the first to demonstrate that regardless of intraspecific genome size variation, Y chromosome has retained its size in S. latifolia. Bioinformatics study of genome composition showed that constancy of Y chromosome size was caused by Y chromosome DNA loss and the female-specific proliferation of recently active dominant retrotransposons. We show that several families of retrotransposons have contributed to genome size variation but not to Y chromosome size change. CONCLUSIONS: Our results suggest that the large Y chromosome of S. latifolia has slowed down or stopped its expansion. Female-specific proliferation of retrotransposons, enlarging the genome with exception of the Y chromosome, was probably caused by silencing of highly active retrotransposons in males and represents an adaptive mechanism to suppress degenerative processes in the haploid stage. Sex specific silencing of transposons might be widespread in plants but hidden in traditional hermaphroditic model plants.

Sex and the flower - developmental aspects of sex chromosome evolution.

Background: The evolution of dioecious plants is occasionally accompanied by the establishment of sex chromosomes: both XY and ZW systems have been found in plants. Structural studies of sex chromosomes are now being followed up by functional studies that are gradually shedding light on the specific genetic and epigenetic processes that shape the development of separate sexes in plants. Scope: This review describes sex determination diversity in plants and the genetic background of dioecy, summarizes recent progress in the investigation of both classical and emerging model dioecious plants and discusses novel findings. The advantages of interspecies hybrids in studies focused on sex determination and the role of epigenetic processes in sexual development are also overviewed. Conclusions: We integrate the genic, genomic and epigenetic levels of sex determination and stress the impact of sex chromosome evolution on structural and functional aspects of plant sexual development. We also discuss the impact of dioecy and sex chromosomes on genome structure and expression.

Impact of repetitive DNA on sex chromosome evolution in plants.

Structurally and functionally diverged sex chromosomes have evolved in many animals as well as in some plants. Sex chromosomes represent a specific genomic region(s) with locally suppressed recombination. As a consequence, repetitive sequences involving transposable elements, tandem repeats (satellites and microsatellites), and organellar DNA accumulate on the Y (W) chromosomes. In this paper, we review the main types of repetitive elements, their gathering on the Y chromosome, and discuss new findings showing that not only accumulation of various repeats in non-recombining regions but also opposite processes form Y chromosome. The aim of this review is also to discuss the mechanisms of repetitive DNA spread involving (retro) transposition, DNA polymerase slippage or unequal crossing-over, as well as modes of repeat removal by ectopic recombination. The intensity of these processes differs in non-recombining region(s) of sex chromosomes when compared to the recombining parts of genome. We also speculate about the relationship between heterochromatinization and the formation of heteromorphic sex chromosomes.

Fully automated pipeline for detection of sex linked genes using RNA-Seq data.

BACKGROUND: Sex chromosomes present a genomic region which to some extent, differs between the genders of a single species. Reliable high-throughput methods for detection of sex chromosomes specific markers are needed, especially in species where genome information is limited. Next generation sequencing (NGS) opens the door for identification of unique sequences or searching for nucleotide polymorphisms between datasets. A combination of classical genetic segregation analysis along with RNA-Seq data can present an ideal tool to map and identify sex chromosome-specific expressed markers. To address this challenge, we established genetic cross of dioecious plant Rumex acetosa and generated RNA-Seq data from both parental generation and male and female offspring. RESULTS: We present a pipeline for detection of sex linked genes based on nucleotide polymorphism analysis. In our approach, tracking of nucleotide polymorphisms is carried out using a cross of preferably distant populations. For this reason, only 4 datasets are needed - reads from high-throughput sequencing platforms for parent generation (mother and father) and F1 generation (male and female progeny). Our pipeline uses custom scripts together with external assembly, mapping and variant calling software. Given the resource-intensive nature of the computation, servers with high capacity are a requirement. Therefore, in order to keep this pipeline easily accessible and reproducible, we implemented it in Galaxy - an open, web-based platform for data-intensive biomedical research. Our tools are present in the Galaxy Tool Shed, from which they can be installed to any local Galaxy instance. As an output of the pipeline, user gets a FASTA file with candidate transcriptionally active sex-linked genes, sorted by their relevance. At the same time, a BAM file with identified genes and alignment of reads is also provided. Thus, polymorphisms following segregation pattern can be easily visualized, which significantly enhances primer design and subsequent steps of wet-lab verification. CONCLUSIONS: Our pipeline presents a simple and freely accessible software tool for identification of sex chromosome linked genes in species without an existing reference genome. Based on combination of genetic crosses and RNA-Seq data, we have designed a high-throughput, cost-effective approach for a broad community of scientists focused on sex chromosome structure and evolution.

Identification of a novel retrotransposon with sex chromosome-specific distribution in Silene latifolia.

Silene latifolia is a dioecious plant species with chromosomal sex determination. Although the evolution of sex chromosomes in S. latifolia has been the subject of numerous studies, a global view of X chromosome structure in this species is still missing. Here, we combine X chromosome microdissection and BAC library screening to isolate new X chromosome-linked sequences. Out of 8 identified BAC clones, only BAC 86M14 showed an X-preferential signal after FISH experiments. Further analysis revealed the existence of the Athila retroelement which is enriched in the X chromosome and nearly absent in the Y chromosome. Based on previous data, the Athila retroelement belongs to the CL3 group of most repetitive sequences in the S. latifolia genome. Structural, transcriptomics and phylogenetic analyses revealed that Athila CL3 represents an old clade in the Athila lineage. We propose a mechanism responsible for Athila CL3 distribution in the S. latifolia genome.

The X chromosome is necessary for somatic development in the dioecious Silene latifolia: cytogenetic and molecular evidence and sequencing of a haploid genome.

Silene latifolia (or white campion) possesses a well-established sex determination system with a dominant Y chromosome in males (the mammalian type). The heteromorphic sex chromosomes X and Y in S. latifolia largely stopped recombination; thus, we can expect a gradual genetic degeneration of the Y chromosome. It is well proven that neither diploid nor polyploid S. latifolia sporophytes can survive without at least one X, so the only life stage possessing the Y as the sole sex chromosome is the male gametophyte (pollen tube), while the female gametophyte seems to be X-dependent. Previous studies on anther-derived plants of this species showed that the obtained plants (largely haploid or dihaploid) were phenotypically and cytologically female. In this paper, we provide molecular evidence for the inviability of plants lacking the X chromosome. Using sex-specific PCR primers, we show that all plantlets and plants derived from anther cultures are female. In studying anther-derived diploid females by sequencing of X-linked markers, we demonstrate that these plants are really homozygous dihaploids. A haploid regenerant plant was sequenced (8× genome coverage) using Illumina technology. Genome data are disposable in the EMBL database as a standard for full genome and X chromosome assembly in this model species. Homozygous dihaploids were back-crossed with males to yield a progeny useful for the study of the evolution of the Y chromosome.

Possible mechanisms responsible for absence of a retrotransposon family on a plant Y chromosome.

Some transposable elements (TEs) show extraordinary variance in abundance along sex chromosomes but the mechanisms responsible for this variance are unknown. Here, we studied Ogre long terminal repeat (LTR) retrotransposons in Silene latifolia, a dioecious plant with evolutionarily young heteromorphic sex chromosomes. Ogre elements are ubiquitous in the S. latifolia genome but surprisingly absent on the Y chromosome. Bacterial artificial chromosome (BAC) library analysis and fluorescence in situ hybridization (FISH) were used to determine Ogre structure and chromosomal localization. Next generation sequencing (NGS) data were analysed to assess the transcription level and abundance of small RNAs. Methylation of Ogres was determined by bisulphite sequencing. Phylogenetic analysis was used to determine mobilization time and selection forces acting on Ogre elements. We characterized three Ogre families ubiquitous in the S. latifolia genome. One family is nearly absent on the Y chromosome despite all the families having similar structures and spreading mechanisms. We showed that Ogre retrotransposons evolved before sex chromosomes appeared but were mobilized after formation of the Y chromosome. Our data suggest that the absence of one Ogre family on the Y chromosome may be caused by 24-nucleotide (24-nt) small RNA-mediated silencing leading to female-specific spreading. Our findings highlight epigenetic silencing mechanisms as potentially crucial factors in sex-specific spreading of some TEs, but other possible mechanisms are also discussed.

Laser Capture Microdissection: From Genomes to Chromosomes, from Complex Tissue to Single-Cell Analysis.

Laser microdissection (LM) is a powerful tool for various molecular analyses providing pure samples for genomic, transcriptomic, and proteomic studies. Cell subgroups, individual cells, or even chromosomes can be separated via laser beam from complex tissues, visualized under the microscope, and used for subsequent molecular analyses. This technique provides information on nucleic acids and proteins, keeping their spatiotemporal information intact. In short, the slide with tissue is placed under the microscope, imaged by a camera onto a computer screen, where the operator selects cells/chromosomes based on morphology or staining and commands the laser beam to cut the specimen following the selected path. Samples are then collected in a tube and subjected to downstream molecular analysis, such as RT-PCR, next-generation sequencing, or immunoassay.

Sperm-dependent asexual species and their role in ecology and evolution.

Sexual reproduction is the primary mode of reproduction in eukaryotes, but some organisms have evolved deviations from classical sex and switched to asexuality. These asexual lineages have sometimes been viewed as evolutionary dead ends, but recent research has revealed their importance in many areas of general biology. Our review explores the understudied, yet important mechanisms by which sperm-dependent asexuals that produce non-recombined gametes but rely on their fertilization, can have a significant impact on the evolution of coexisting sexual species and ecosystems. These impacts are concentrated around three major fields. Firstly, sperm-dependent asexuals can potentially impact the gene pool of coexisting sexual species by either restricting their population sizes or by providing bridges for interspecific gene flow whose type and consequences substantially differ from gene flow mechanisms expected under sexual reproduction. Secondly, they may impact on sexuals' diversification rates either directly, by serving as stepping-stones in speciation, or indirectly, by promoting the formation of pre- and postzygotic reproduction barriers among nascent species. Thirdly, they can potentially impact on spatial distribution of species, via direct or indirect (apparent) types of competition and Allee effects. For each such mechanism, we provide empirical examples of how natural sperm-dependent asexuals impact the evolution of their sexual counterparts. In particular, we highlight that these broad effects may last beyond the tenure of the individual asexual lineages causing them, which challenges the traditional perception that asexual lineages are short-lived evolutionary dead ends and minor sideshows. Our review also proposes new research directions to incorporate the aforementioned impacts of sperm-dependent asexuals. These research directions will ultimately enhance our understanding of the evolution of genomes and biological interactions in general.