Proteome and Interactome Linked to Metabolism, Genetic Information Processing, and Abiotic Stress in Gametophytes of Two Woodferns.

Ferns and lycophytes have received scant molecular attention in comparison to angiosperms. The advent of high-throughput technologies allowed an advance towards a greater knowledge of their elusive genomes. In this work, proteomic analyses of heart-shaped gametophytes of two ferns were performed: the apomictic Dryopteris affinis ssp. affinis and its sexual relative Dryopteris oreades. In total, a set of 218 proteins shared by these two gametophytes were analyzed using the STRING database, and their proteome associated with metabolism, genetic information processing, and responses to abiotic stress is discussed. Specifically, we report proteins involved in the metabolism of carbohydrates, lipids, and nucleotides, the biosynthesis of amino acids and secondary compounds, energy, oxide-reduction, transcription, translation, protein folding, sorting and degradation, and responses to abiotic stresses. The interactome of this set of proteins represents a total network composed of 218 nodes and 1792 interactions, obtained mostly from databases and text mining. The interactions among the identified proteins of the ferns D. affinis and D. oreades, together with the description of their biological functions, might contribute to a better understanding of the function and development of ferns as well as fill knowledge gaps in plant evolution.

The Shared Proteome of the Apomictic Fern Dryopteris affinis ssp. affinis and Its Sexual Relative Dryopteris oreades.

Ferns are a diverse evolutionary lineage, sister to the seed plants, which is of great ecological importance and has a high biotechnological potential. Fern gametophytes represent one of the simplest autotrophic, multicellular plant forms and show several experimental advantages, including a simple and space-efficient in vitro culture system. However, the molecular basis of fern growth and development has hardly been studied. Here, we report on a proteomic study that identified 417 proteins shared by gametophytes of the apogamous fern Dryopteris affinis ssp. affinis and its sexual relative Dryopteris oreades. Most proteins are predicted to localize to the cytoplasm, the chloroplast, or the nucleus, and are linked to enzymatic, binding, and structural activities. A subset of 145 proteins are involved in growth, reproduction, phytohormone signaling and biosynthesis, and gene expression, including homologs of SHEPHERD (SHD), HEAT SHOCK PROTEIN 90-5 (CR88), TRP4, BOBBER 1 (BOB1), FLAVONE 3'-O-METHYLTRANSFERASE 1 (OMT1), ZEAXANTHIN EPOXIDASE (ABA1), GLUTAMATE DESCARBOXYLASE 1 (GAD), and dsRNA-BINDING DOMAIN-LIKE SUPERFAMILY PROTEIN (HLY1). Nearly 25% of the annotated proteins are associated with responses to biotic and abiotic stimuli. As for biotic stress, the proteins PROTEIN SGT1 HOMOLOG B (SGT1B), SUPPRESSOR OF SA INSENSITIVE2 (SSI2), PHOSPHOLIPASE D ALPHA 1 (PLDALPHA1), SERINE/THREONINE-PROTEIN KINASE SRK2E (OST1), ACYL CARRIER PROTEIN 4 (ACP4), and NONHOST RESISTANCE TO P. S. PHASEOLICOLA1 (GLPK) are worth mentioning. Regarding abiotic stimuli, we found proteins associated with oxidative stress: SUPEROXIDE DISMUTASE[CU-ZN] 1 (CSD1), and GLUTATHIONE S-TRANSFERASE U19 (GSTU19), light intensity SERINE HYDROXYMETHYLTRANSFERASE 1 (SHM1) and UBIQUITIN-CONJUGATING ENZYME E2 35 (UBC35), salt and heavy metal stress included MITOCHONDRIAL PHOSPHATE CARRIER PROTEIN 3 (PHT3;1), as well as drought and thermotolerance: LEA7, DEAD-BOX ATP-DEPENDENT RNA HELICASE 38 (LOS4), and abundant heat-shock proteins and other chaperones. In addition, we identified interactomes using the STRING platform, revealing protein-protein associations obtained from co-expression, co-occurrence, text mining, homology, databases, and experimental datasets. By focusing on ferns, this proteomic study increases our knowledge on plant development and evolution, and may inspire future applications in crop species.

Sexual and Apogamous Species of Woodferns Show Different Protein and Phytohormone Profiles.

The gametophyte of ferns reproduces either by sexual or asexual means. In the latter, apogamy represents a peculiar case of apomixis, in which an embryo is formed from somatic cells. A proteomic and physiological approach was applied to the apogamous fern Dryopteris affinis ssp. affinis and its sexual relative D. oreades. The proteomic analysis compared apogamous vs. female gametophytes, whereas the phytohormone study included, in addition to females, three apogamous stages (filamentous, spatulate, and cordate). The proteomic profiles revealed a total of 879 proteins and, after annotation, different regulation was found in 206 proteins of D. affinis and 166 of its sexual counterpart. The proteins upregulated in D. affinis are mostly associated to protein metabolism (including folding, transport, and proteolysis), ribosome biogenesis, gene expression and translation, while in the sexual counterpart, they account largely for starch and sucrose metabolism, generation of energy and photosynthesis. Likewise, ultra-performance liquid chromatography-tandem spectrometry (UHPLC-MS/MS) was used to assess the levels of indol-3-acetic acid (IAA); the cytokinins: 6-benzylaminopurine (BA), trans-Zeatine (Z), trans-Zeatin riboside (ZR), dyhidrozeatine (DHZ), dyhidrozeatin riboside (DHZR), isopentenyl adenine (iP), isopentenyl adenosine (iPR), abscisic acid (ABA), the gibberellins GA3 and GA4, salicylic acid (SA), and the brassinosteroids: brassinolide (BL) and castasterone (CS). IAA, the cytokinins Z, ZR, iPR, the gibberellin GA4, the brassinosteoids castasterone, and ABA accumulated more in the sexual gametophyte than in the apogamous one. When comparing the three apogamous stages, BA and SA peaked in filamentous, GA3 and BL in spatulate and DHRZ in cordate gametophytes. The results point to the existence of large metabolic differences between apogamous and sexual gametophytes, and invite to consider the fern gametophyte as a good experimental system to deepen our understanding of plant reproduction.

The Polycomb group protein MEDEA controls cell proliferation and embryonic patterning in Arabidopsis.

Establishing the embryonic body plan of multicellular organisms relies on precisely orchestrated cell divisions coupled with pattern formation, which, in animals, are regulated by Polycomb group (PcG) proteins. The conserved Polycomb Repressive Complex 2 (PRC2) mediates H3K27 trimethylation and comes in different flavors in Arabidopsis. The PRC2 catalytic subunit MEDEA is required for seed development; however, a role for PRC2 in embryonic patterning has been dismissed. Here, we demonstrate that embryos derived from medea eggs abort because MEDEA is required for patterning and cell lineage determination in the early embryo. Similar to PcG proteins in mammals, MEDEA regulates embryonic patterning and growth by controlling cell-cycle progression through repression of CYCD1;1, which encodes a core cell-cycle component. Thus, Arabidopsis embryogenesis is epigenetically regulated by PcG proteins, revealing that the PRC2-dependent modulation of cell-cycle progression was independently recruited to control embryonic cell proliferation and patterning in animals and plants.

Differential gene expression profiling of one- and two-dimensional apogamous gametophytes of the fern Dryopteris affinis ssp. affinis.

Apomixis was originally defined as the replacement of sexual reproduction by an asexual process that does not involve fertilization but, in angiosperms, it is often used in the more restricted sense of asexual reproduction through seeds. In ferns, apomixis combines the production of unreduced spores (diplospory) and the formation of sporophytes from somatic cells of the prothallium (apogamy). The genes that control the onset of apogamy in ferns are largely unknown. In this study, we describe the gametophyte transcriptome of the apogamous fern Dryopteris affinis ssp. affinis using an RNA-Seq approach to compare the gene expression profiles of one- and two-dimensional gametophytes, the latter containing apogamic centers. After collapsing highly similar de novo transcripts, we obtained 166,191 unigenes, of which 30% could be annotated using public databases. Multiple quality metrics indicate a good quality of the de novo transcriptome with a low level of fragmentation. Our data show a total of 10,679 genes (6% of all genes) to be differentially expressed between gametophytes of filamentous (one-dimensional) and prothallial (two-dimensional) architecture. 6,110 genes were up-regulated in two-dimensional relative to one-dimensional gametophytes, some of which are implicated in the regulation of meristem growth, auxin signaling, reproduction, and sucrose metabolism. 4,570 genes were down-regulated in two-dimensional versus one-dimensional gametophytes, which are enriched in stimulus and defense genes, as well as genes involved in epigenetic gene regulation and ubiquitin degradation. Our results provide insights into free-living gametophyte development, focusing on the filamentous-to-prothallus growth transition, and provide a useful resource for further investigations of asexual reproduction.

Contribution of epigenetic variation to adaptation in Arabidopsis.

In plants, transgenerational inheritance of some epialleles has been demonstrated but it remains controversial whether epigenetic variation is subject to selection and contributes to adaptation. Simulating selection in a rapidly changing environment, we compare phenotypic traits and epigenetic variation between Arabidopsis thaliana populations grown for five generations under selection and their genetically nearly identical ancestors. Selected populations of two distinct genotypes show significant differences in flowering time and plant architecture, which are maintained for at least 2-3 generations in the absence of selection. While we cannot detect consistent genetic changes, we observe a reduction of epigenetic diversity and changes in the methylation state of about 50,000 cytosines, some of which are associated with phenotypic changes. Thus, we propose that epigenetic variation is subject to selection and can contribute to rapid adaptive responses, although the extent to which epigenetics plays a role in adaptation is still unclear.

RALF4/19 peptides interact with LRX proteins to control pollen tube growth in Arabidopsis.

The communication of changes in the extracellular matrix to the interior of the cell is crucial for a cell's function. The extracellular peptides of the RAPID ALKALINIZATION FACTOR (RALF) family have been identified as ligands of receptor-like kinases of the CrRLK1L subclass, but the exact mechanism of their perception is unclear. We found that Arabidopsis RALF4 and RALF19 redundantly regulate pollen tube integrity and growth, and that their function depends on pollen-expressed proteins of the LEUCINE-RICH REPEAT EXTENSIN (LRX) family, which play a role in cell wall development but whose mode of action is not understood. The LRX proteins interact with RALFs, monitoring cell wall changes, which are communicated to the interior of the pollen tube via the CrRLK1L pathway to sustain normal growth.

Proteogenomic Analysis Greatly Expands the Identification of Proteins Related to Reproduction in the Apogamous Fern Dryopteris affinis ssp. affinis.

Performing proteomic studies on non-model organisms with little or no genomic information is still difficult. However, many specific processes and biochemical pathways occur only in species that are poorly characterized at the genomic level. For example, many plants can reproduce both sexually and asexually, the first one allowing the generation of new genotypes and the latter their fixation. Thus, both modes of reproduction are of great agronomic value. However, the molecular basis of asexual reproduction is not well understood in any plant. In ferns, it combines the production of unreduced spores (diplospory) and the formation of sporophytes from somatic cells (apogamy). To set the basis to study these processes, we performed transcriptomics by next-generation sequencing (NGS) and shotgun proteomics by tandem mass spectrometry in the apogamous fern D. affinis ssp. affinis. For protein identification we used the public viridiplantae database (VPDB) to identify orthologous proteins from other plant species and new transcriptomics data to generate a "species-specific transcriptome database" (SSTDB). In total 1,397 protein clusters with 5,865 unique peptide sequences were identified (13 decoy proteins out of 1,410, protFDR 0.93% on protein cluster level). We show that using the SSTDB for protein identification increases the number of identified peptides almost four times compared to using only the publically available VPDB. We identified homologs of proteins involved in reproduction of higher plants, including proteins with a potential role in apogamy. With the increasing availability of genomic data from non-model species, similar proteogenomics approaches will improve the sensitivity in protein identification for species only distantly related to models.

Starch Turnover and Metabolism during Flower and Early Embryo Development.

The accumulation of starch within photosynthetic tissues and within dedicated storage organs has been characterized extensively in many species, and a function in buffering carbon availability or in fueling later growth phases, respectively, has been proposed. However, developmentally regulated starch turnover within heterotrophic tissues other than dedicated storage organs is poorly characterized, and its function is not well understood. Here, we report on the characterization of starch turnover during flower, early embryo, and silique development in Arabidopsis (Arabidopsis thaliana) using a combined clearing-staining technique on whole-mount tissue. Besides the two previously documented waves of transient starch accumulation in the stamen envelope, occurring during meiosis and pollen mitosis I, we identified a novel, third wave of starch amylogenesis/amylolysis during the last stages of stamen development. To gain insights into the underlying molecular mechanisms, we analyzed publicly available microarray data, which revealed a developmentally coordinated expression of carbohydrate transport and metabolism genes during these waves of transient starch accumulation. Based on this analysis, we characterized starch dynamics in mutants affecting hexose phosphate metabolism and translocation, and identified the Glc-6-phosphate/phosphate antiporter GPT1 as the putative translocator of Glc-6-phosphate for starch biosynthesis in reproductive tissues. Based on these results, we propose a model of starch synthesis within the pollen grain and discuss the nutrient transport route feeding the embryo within the developing seed.

A subunit of the oligosaccharyltransferase complex is required for interspecific gametophyte recognition in Arabidopsis.

Species-specific gamete recognition is a key premise to ensure reproductive success and the maintenance of species boundaries. During plant pollen tube (PT) reception, gametophyte interactions likely allow the species-specific recognition of signals from the PT (male gametophyte) by the embryo sac (female gametophyte), resulting in PT rupture, sperm release, and double fertilization. This process is impaired in interspecific crosses between Arabidopsis thaliana and related species, leading to PT overgrowth and a failure to deliver the sperm cells. Here we show that ARTUMES (ARU) specifically regulates the recognition of interspecific PTs in A. thaliana. ARU, identified in a genome-wide association study (GWAS), exclusively influences interspecific--but not intraspecific--gametophyte interactions. ARU encodes the OST3/6 subunit of the oligosaccharyltransferase complex conferring protein N-glycosylation. Our results suggest that glycosylation patterns of cell surface proteins may represent an important mechanism of gametophyte recognition and thus speciation.

Efficient and rapid isolation of early-stage embryos from Arabidopsis thaliana seeds.

In flowering plants, the embryo develops within a nourishing tissue - the endosperm - surrounded by the maternal seed integuments (or seed coat). As a consequence, the isolation of plant embryos at early stages (1 cell to globular stage) is technically challenging due to their relative inaccessibility. Efficient manual dissection at early stages is strongly impaired by the small size of young Arabidopsis seeds and the adhesiveness of the embryo to the surrounding tissues. Here, we describe a method that allows the efficient isolation of young Arabidopsis embryos, yielding up to 40 embryos in 1 hr to 4 hr, depending on the downstream application. Embryos are released into isolation buffer by slightly crushing 250-750 seeds with a plastic pestle in an Eppendorf tube. A glass microcapillary attached to either a standard laboratory pipette (via a rubber tube) or a hydraulically controlled microinjector is used to collect embryos from droplets placed on a multi-well slide on an inverted light microscope. The technical skills required are simple and easily transferable, and the basic setup does not require costly equipment. Collected embryos are suitable for a variety of downstream applications such as RT-PCR, RNA sequencing, DNA methylation analyses, fluorescence in situ hybridization (FISH), immunostaining, and reporter gene assays.

TAF13 interacts with PRC2 members and is essential for Arabidopsis seed development.

TBP-Associated Factors (TAFs) are components of complexes like TFIID, TFTC, SAGA/STAGA and SMAT that are important for the activation of transcription, either by establishing the basic transcription machinery or by facilitating histone acetylation. However, in Drosophila embryos several TAFs were shown to be associated with the Polycomb Repressive Complex 1 (PRC1), even though the role of this interaction remains unclear. Here we show that in Arabidopsis TAF13 interacts with MEDEA and SWINGER, both members of a plant variant of Polycomb Repressive Complex 2 (PRC2). PRC2 variants play important roles during the plant life cycle, including seed development. The taf13 mutation causes seed defects, showing embryo arrest at the 8-16 cell stage and over-proliferation of the endosperm in the chalazal region, which is typical for Arabidopsis PRC2 mutants. Our data suggest that TAF13 functions together with PRC2 in transcriptional regulation during seed development.

The Polycomb group protein MEDEA and the DNA methyltransferase MET1 interact to repress autonomous endosperm development in Arabidopsis.

In flowering plants, double fertilization of the female gametes, the egg and the central cell, initiates seed development to give rise to a diploid embryo and the triploid endosperm. In the absence of fertilization, the FERTILIZATION-INDEPENDENT SEED Polycomb Repressive Complex 2 (FIS-PRC2) represses this developmental process by histone methylation of certain target genes. The FERTILIZATION-INDEPENDENT SEED (FIS) class genes MEDEA (MEA) and FERTILIZATION-INDEPENDENT ENDOSPERM (FIE) encode two of the core components of this complex. In addition, DNA methylation establishes and maintains the repression of gene activity, for instance via DNA METHYLTRANSFERASE1 (MET1), which maintains methylation of symmetric CpG residues. Here, we demonstrate that Arabidopsis MET1 interacts with MEA in vitro and in a yeast two-hybrid assay, similar to the previously identified interaction of the mammalian homologues DNMT1 and EZH2. MET1 and MEA share overlapping expression patterns in reproductive tissues before and after fertilization, a prerequisite for an interaction in vivo. Importantly, a much higher percentage of central cells initiate endosperm development in the absence of fertilization in mea-1/MEA; met1-3/MET1 as compared to mea-1/MEA mutant plants. In addition, DNA methylation at the PHERES1 and MEA loci, imprinted target genes of the FIS-PRC2, was affected in the mea-1 mutant compared with wild-type embryos. In conclusion, our data suggest a mechanistic link between two major epigenetic pathways involved in histone and DNA methylation in plants by physical interaction of MET1 with the FIS-PRC2 core component MEA. This concerted action is relevant for the repression of seed development in the absence of fertilization.

Identification of a DNA methylation-independent imprinting control region at the Arabidopsis MEDEA locus.

Genomic imprinting is exclusive to mammals and seed plants and refers to parent-of-origin-dependent, differential transcription. As previously shown in mammals, studies in Arabidopsis have implicated DNA methylation as an important hallmark of imprinting. The current model suggests that maternally expressed imprinted genes, such as MEDEA (MEA), are activated by the DNA glycosylase DEMETER (DME), which removes DNA methylation established by the DNA methyltransferase MET1. We report the systematic functional dissection of the MEA cis-regulatory region, resulting in the identification of a 200-bp fragment that is necessary and sufficient to mediate MEA activation and imprinted expression, thus containing the imprinting control region (ICR). Notably, imprinted MEA expression mediated by this ICR is independent of DME and MET1, consistent with the lack of any significant DNA methylation in this region. This is the first example of an ICR without differential DNA methylation, suggesting that factors other than DME and MET1 are required for imprinting at the MEA locus.

Stearoyl-acyl carrier protein desaturases are associated with floral isolation in sexually deceptive orchids.

The orchids Ophrys sphegodes and O. exaltata are reproductively isolated from each other by the attraction of two different, highly specific pollinator species. For pollinator attraction, flowers chemically mimic the pollinators' sex pheromones, the key components of which are alkenes with different double-bond positions. This study identifies genes likely involved in alkene biosynthesis, encoding stearoyl-acyl carrier protein (ACP) desaturase (SAD) homologs. The expression of two isoforms, SAD1 and SAD2, is flower-specific and broadly parallels alkene production during flower development. SAD2 shows a significant association with alkene production, and in vitro assays show that O. sphegodes SAD2 has activity both as an 18:0-ACP Δ(9) and a 16:0-ACP Δ(4) desaturase. Downstream metabolism of the SAD2 reaction products would give rise to alkenes with double-bonds at position 9 or position 12, matching double-bond positions observed in alkenes in the odor bouquet of O. sphegodes. SAD1 and SAD2 show evidence of purifying selection before, and positive or relaxed purifying selection after gene duplication. By contributing to the production of species-specific alkene bouquets, SAD2 is suggested to contribute to differential pollinator attraction and reproductive isolation among these species. Taken together, these data are consistent with the hypothesis that SAD2 is a florally expressed barrier gene of large phenotypic effect and, possibly, a genic target of pollinator-mediated selection.

The FERONIA receptor-like kinase mediates male-female interactions during pollen tube reception.

In flowering plants, signaling between the male pollen tube and the synergid cells of the female gametophyte is required for fertilization. In the Arabidopsis thaliana mutant feronia (fer), fertilization is impaired; the pollen tube fails to arrest and thus continues to grow inside the female gametophyte. FER encodes a synergid-expressed, plasma membrane-localized receptor-like kinase. We found that the FER protein accumulates asymmetrically in the synergid membrane at the filiform apparatus. Interspecific crosses using pollen from Arabidopsis lyrata and Cardamine flexuosa on A. thaliana stigmas resulted in a fer-like phenotype that correlates with sequence divergence in the extracellular domain of FER. Our findings show that the female control of pollen tube reception is based on a FER-dependent signaling pathway, which may play a role in reproductive isolation barriers.

Positive darwinian selection at the imprinted MEDEA locus in plants.

In mammals and seed plants, a subset of genes is regulated by genomic imprinting where an allele's activity depends on its parental origin. The parental conflict theory suggests that genomic imprinting evolved after the emergence of an embryo-nourishing tissue (placenta and endosperm), resulting in an intragenomic parental conflict over the allocation of nutrients from mother to offspring. It was predicted that imprinted genes, which arose through antagonistic co-evolution driven by a parental conflict, should be subject to positive darwinian selection. Here we show that the imprinted plant gene MEDEA (MEA), which is essential for seed development, originated during a whole-genome duplication 35 to 85 million years ago. After duplication, MEA underwent positive darwinian selection consistent with neo-functionalization and the parental conflict theory. MEA continues to evolve rapidly in the out-crossing species Arabidopsis lyrata but not in the self-fertilizing species Arabidopsis thaliana, where parental conflicts are reduced. The paralogue of MEA, SWINGER (SWN; also called EZA1), is not imprinted and evolved under strong purifying selection because it probably retained the ancestral function of the common precursor gene. The evolution of MEA suggests a late origin of genomic imprinting within the Brassicaceae, whereas imprinting is thought to have originated early within the mammalian lineage.

Dynamic regulatory interactions of Polycomb group genes: MEDEA autoregulation is required for imprinted gene expression in Arabidopsis.

The imprinted Arabidopsis Polycomb group (PcG) gene MEDEA (MEA), which is homologous to Enhancer of Zeste [E(Z)], is maternally required for normal seed development. Here we show that, unlike known mammalian imprinted genes, MEA regulates its own imprinted expression: It down-regulates the maternal allele around fertilization and maintains the paternal allele silent later during seed development. Autorepression of the maternal MEA allele is direct and independent of the MEA-FIE (FERTILIZATION-INDEPENDENT ENDOSPERM) PcG complex, which is similar to the E(Z)-ESC (Extra sex combs) complex of animals, suggesting a novel mechanism. A complex network of cross-regulatory interactions among the other known members of the MEA-FIE PcG complex implies distinct functions that are dynamically regulated during reproduction.

The RPN1 subunit of the 26S proteasome in Arabidopsis is essential for embryogenesis.

The 26S proteasome plays a central role in the degradation of regulatory proteins involved in a variety of developmental processes. It consists of two multisubunit protein complexes: the proteolytic core protease and the regulatory particle (RP). The function of most RP subunits is poorly understood. Here, we describe mutants in the Arabidopsis thaliana RPN1 subunit, which is encoded by two paralogous genes, RPN1a and RPN1b. Disruption of RPN1a caused embryo lethality, while RPN1b mutants showed no obvious abnormal phenotype. Embryos homozygous for rpn1a arrested at the globular stage with defects in the formation of the embryonic root, the protoderm, and procambium. Cyclin B1 protein was not degraded in these embryos, consistent with cell division defects. Double mutant plants (rpn1a/RPN1a rpn1b/rpn1b) produced embryos with a phenotype indistinguishable from that of the rpn1a single mutant. Thus, despite their largely overlapping expression patterns in flowers and developing seeds, the two isoforms do not share redundant functions during gametogenesis and embryogenesis. However, complementation of the rpn1a mutation with the coding region of RPN1b expressed under the control of the RPN1a promoter indicates that the two RPN1 isoforms are functionally equivalent. Overall, our data indicate that RPN1 activity is essential during embryogenesis, where it might participate in the destruction of a specific set of protein substrates.

The Arabidopsis thaliana MEDEA Polycomb group protein controls expression of PHERES1 by parental imprinting.

The maternally expressed Arabidopsis thaliana Polycomb group protein MEDEA (MEA) controls expression of the MADS-box gene PHERES1 (PHE1). Here, we show that PHE1 is mainly paternally expressed but maternally repressed and that this maternal repression of PHE1 breaks down in seeds lacking maternal MEA activity. Because Polycomb group proteins control parental imprinting in mammals as well, the independent recruitment of similar protein machineries for the imprinting of genes is a notable example of convergent evolution.