Events Of Sexual Reproduction Research Articles

Deep learning insights into the architecture of the mammalian egg-sperm fusion synapse

A crucial event in sexual reproduction is when haploid sperm and egg fuse to form a new diploid organism at fertilization. In mammals, direct interaction between egg JUNO and sperm IZUMO1 mediates gamete membrane adhesion, yet their role in fusion remains enigmatic. We used AlphaFold to predict the structure of other extracellular proteins essential for fertilization to determine if they could form a complex that may mediate fusion. We first identified TMEM81, whose gene is expressed by mouse and human spermatids, as a protein having structural homologies with both IZUMO1 and another sperm molecule essential for gamete fusion, SPACA6. Using a set of proteins known to be important for fertilization and TMEM81, we then systematically searched for predicted binary interactions using an unguided approach and identified a pentameric complex involving sperm IZUMO1, SPACA6, TMEM81 and egg JUNO, CD9. This complex is structurally consistent with both the expected topology on opposing gamete membranes and the location of predicted N-glycans not modeled by AlphaFold-Multimer, suggesting that its components could organize into a synapse-like assembly at the point of fusion. Finally, the structural modeling approach described here could be more generally useful to gain insights into transient protein complexes difficult to detect experimentally.

Deep learning insights into the architecture of the mammalian egg-sperm fusion synapse.

A crucial event in sexual reproduction is when haploid sperm and egg fuse to form a new diploid organism at fertilization. In mammals, direct interaction between egg JUNO and sperm IZUMO1 mediates gamete membrane adhesion, yet their role in fusion remains enigmatic. We used AlphaFold to predict the structure of other extracellular proteins essential for fertilization to determine if they could form a complex that may mediate fusion. We first identified TMEM81, whose gene is expressed by mouse and human spermatids, as a protein having structural homologies with both IZUMO1 and another sperm molecule essential for gamete fusion, SPACA6. Using a set of proteins known to be important for fertilization and TMEM81, we then systematically searched for predicted binary interactions using an unguided approach and identified a pentameric complex involving sperm IZUMO1, SPACA6, TMEM81 and egg JUNO, CD9. This complex is structurally consistent with both the expected topology on opposing gamete membranes and the location of predicted N-glycans not modeled by AlphaFold-Multimer, suggesting that its components could organize into a synapse-like assembly at the point of fusion. Finally, the structural modeling approach described here could be more generally useful to gain insights into transient protein complexes difficult to detect experimentally.

Arbuscular mycorrhizal fungi heterokaryons have two nuclear populations with distinct roles in host-plant interactions.

Arbuscular mycorrhizal fungi (AMF) are prominent root symbionts that can carry thousands of nuclei deriving from two parental strains in a large syncytium. These co-existing genomes can also vary in abundance with changing environmental conditions. Here we assemble the nuclear genomes of all four publicly available AMF heterokaryons using PacBio high-fidelity and Hi-C sequencing. We find that the two co-existing genomes of these strains are phylogenetically related but differ in structure, content and epigenetics. We confirm that AMF heterokaryon genomes vary in relative abundance across conditions and show this can lead to nucleus-specific differences in expression during interactions with plants. Population analyses also reveal signatures of genetic exchange indicative of past events of sexual reproduction in these strains. This work uncovers the origin and contribution of two nuclear genomes in AMF heterokaryons and opens avenues for the improvement and environmental application of these strains.

The origination events of gametic sexual reproduction and anisogamy

The evolution of gametic sex (meiosis and fertilization) and subsequent transition from isogamy (fusion between two equal-sized gametes) to anisogamy (dimorphism into eggs and sperm, namely, females and males) is one of the largest enigmas of evolutionary biology. Meiosis entails genome-dilution cost and anisogamy entails male-production cost. Despite much progress has been made for the maintenance mechanisms of sex, its origination events under such “twofold cost of sex” are still unsolved. Here, we posit two hypothetical scenarios as follows: the “Seesaw Effect” hypothesizes that automictic selfing between isogametes effectively purged deleterious mutations from an organism’s lineage and simultaneously fixed the sex-controlling allele and all other loci (no genome-dilution cost raised). The high relatedness among homoeologous cell colonies led to multicellularization. The “inflated isogamy” hypothesizes that multicellularity increased the reproductive investment of both mates, resulting in excessively large isogametes. This redundancy induced cheating of one sex (evolving to male) to reduce gamete size. However, the other sex (evolving to female) allowed this cheat because her cost did not change. Therefore, anisogamy originated as a kind of commensalism but turned into beneficial for females because it solved the gamete limitation problem inherent to isogamy. Thus, smooth transition to anisogamy had been attained.

C11orf94/Frey is a key regulator for male fertility by controlling Izumo1 complex assembly.

Although gamete fusion represents the central event in sexual reproduction, the required protein machinery is poorly defined. In sperm cells, Izumo1 and several Izumo1-associated proteins play an essential role for this process. However, so far, the mechanisms underlying transport and maturation of Izumo1 and its incorporation into high molecular weight complexes are incompletely defined. Here, we provide a detailed characterization of the C11orf94 protein, which we rename Frey, which provides a platform for the assembly of Izumo1 complexes. By retaining Izumo1 in the endoplasmic reticulum, Frey facilitates its incorporation into high molecular weight complexes. To fulfill its function, the unstable Frey protein is stabilized within the catalytic center of an intramembrane protease. Loss of Frey results in reduced assembly of Izumo1 complexes and male infertility due to impaired gamete fusion. Collectively, these findings provide mechanistic insights into the early biogenesis and functional relevance of Izumo1 complexes.

Cytidinediphosphate diacylglycerol synthase-Mediated phosphatidic acid metabolism is crucial for early embryonic development of Arabidopsis.

Embryonic development is a key developmental event in plant sexual reproduction; however, regulatory networks of plant early embryonic development, particularly the effects and functional mechanisms of phospholipid molecules are still unknown due to the limitation of sample collection and analysis. We innovatively applied the microspore-derived in vitro embryogenesis of Brassica napus and revealed the dynamics of phospholipid molecules, especially phosphatidic acid (PA, an important second messenger that plays an important role in plant growth, development, and stress responses), at different embryonic developmental stages by using a lipidomics approach. Further analysis of Arabidopsis mutants deficiency of CDS1 and CDS2 (cytidinediphosphate diacylglycerol synthase, key protein in PA metabolism) revealed the delayed embryonic development from the proembryo stage, indicating the crucial effect of CDS and PA metabolism in early embryonic development. Decreased auxin level and disturbed polar localization of auxin efflux carrier PIN1 implicate that CDS-mediated PA metabolism may regulate early embryogenesis through modulating auxin transport and distribution. These results demonstrate the dynamics and importance of phospholipid molecules during embryo development, and provide informative clues to elucidate the regulatory network of embryogenesis.

Leishmania braziliensis causing human disease in Northeast Brazil presents loci with genotypes in long-term equilibrium.

Leishmaniases are neglected tropical diseases that inflict great burden to poor areas of the globe. Intense research has aimed to identify parasite genetic signatures predictive of infection outcomes. Consistency of diagnostic tools based on these markers would greatly benefit from accurate understanding of Leishmania spp. population genetics. We explored two chromosomal loci to characterize a population of L. braziliensis causing human disease in Northeast Brazil. Two temporally distinct samples of L. braziliensis were obtained from patients attending the leishmaniasis clinic at the village of Corte de Pedra: (2008-2011) primary sample, N = 120; (1999-2001) validation sample, N = 35. Parasites were genotyped by Sanger's sequencing of two 600 base pairs loci starting at nucleotide positions 3,074 and 425,451 of chromosomes 24 and 28, respectively. Genotypes based on haplotypes of biallelic positions in each locus were tested for several population genetic parameters as well as for geographic clustering within the region. Ample geographic overlap of genotypes at the two loci was observed as indicated by non-significant Cusick and Edward's comparisons. No linkage disequilibrium was detected among combinations of haplotypes for both parasite samples. Homozygous and heterozygous genotypes displayed Hardy-Weinberg equilibrium (HWE) at both loci in the two samples when straight observed and expected counts were compared by Chi-square (p>0.5). However, Bayesian statistics using one million Monte-Carlo randomizations disclosed a less robust HWE for chromosome 24 genotypes, particularly in the primary sample (p = 0.04). Fixation indices (Fst) were consistently lower than 0.05 among individuals of the two samples at both tested loci, and no intra-populational structuralization could be detected using STRUCTURE software. These findings suggest that L. braziliensis can maintain stable populations in foci of human leishmaniasis and are capable of robust genetic recombination possibly due to events of sexual reproduction during the parasite's lifecycle.

The Role of Sperm Proteins IZUMO1 and TMEM95 in Mammalian Fertilization: A Systematic Review.

Gamete membrane fusion is a critical cellular event in sexual reproduction. In addition, the generation of knockout models has provided a powerful tool for testing the functional relevance of proteins thought to be involved in mammalian fertilization, suggesting IZUMO1 and TMEM95 (transmembrane protein 95) as essential proteins. However, the molecular mechanisms underlying the process remain largely unknown. Therefore, the aim of this study was to summarize the current knowledge about IZUMO1 and TMEM95 during mammalian fertilization. Hence, three distinct databases were consulted—PubMed, Scopus and Web of Science—using single keywords. As a result, a total of 429 articles were identified. Based on both inclusion and exclusion criteria, the final number of articles included in this study was 103. The results showed that IZUMO1 is mostly studied in rodents whereas TMEM95 is studied primarily in bovines. Despite the research, the topological localization of IZUMO1 remains controversial. IZUMO1 may be involved in organizing or stabilizing a multiprotein complex essential for the membrane fusion in which TMEM95 could act as a fusogen due to its possible interaction with IZUMO1. Overall, the expression of these two proteins is not sufficient for sperm–oocyte fusion; therefore, other molecules must be involved in the membrane fusion process.

Microsatellite Analysis Revealing High Genetic Diversity of the Chestnut Blight Fungus in South Tyrol (Northern Italy)

Cryphonectria parasitica, which causes chestnut blight, is one of the most important pathogens of forest trees. In Europe, mycovirus-mediated biocontrol is the most efficient method to control the disease but can be impeded by the lack of information about the population structure of the fungus within a region. In particular, sexual reproduction and the new introduction of the pathogen can complicate biocontrol strategies. For this reason, this study aimed to determine the population structure of C. parasitica, which causes chestnut blight, in the northern Italian region of South Tyrol, using eleven multilocus microsatellite markers. Fifty-one haplotypes were found across South Tyrol, belonging to three divergent clusters. Recombinant genotypes demonstrated that sexual reproduction occurs across the different clusters. The most dominant genotypes in the region were also the most dominant in neighboring areas, such as Switzerland, northern Italy and France. All of the clusters from South Tyrol were related to the Italian genotype pool and are thought to have been introduced from northern Italian and other European populations due to naturally occurring gene flow or human-mediated introduction. At least three separate introduction events of C. parasitica might have happened in South Tyrol that could be separated by time. This study demonstrated a high genetic diversity of C. parasitica in South Tyrol and helped to shed light on the sexual reproduction and introduction events in the local populations.

Evidence of Sexual Reproduction Events in the Dagger Nematode Xiphinema index in Grapevine Resistance Experiments Under Controlled Conditions.

The dagger nematode Xiphinema index has a major economic impact because of its transmission of Grapevine fanleaf virus to grapevines. This vector nematode, which was introduced into Western countries from the Middle East together with the domesticated grapevine, mostly reproduces by meiotic parthenogenesis, but microsatellite multilocus genotype (MLG) analysis has revealed the occurrence of rare sexual reproduction events in field conditions. In a previous 6-year study under controlled conditions, we evaluated the durability of resistance to X. index in accessions derived from a muscadine resistance source and reference accessions. In this previous study, we used an equal-proportion mixture of four lines (from Spain, Italy, Greece, and Iran) representative of X. index diversity as the inoculum, and we collected random samples in 3-, 4-, 5-, and 6-year-old vines. Here, we genotyped the individuals from these samples using the MLG technique, and we analyzed the changes in line frequency and the occurrence of sexual reproduction events between lines over time. The nematode lines differed in aggressiveness and hybrids between lines were detected at a low, but apparently increasing rate. Hybridization events were recovered in all accessions, regardless of resistance status and propagation type. Finally, our data provide the first evidence of sexual reproduction in the nematode X. index under controlled conditions.

Sex-Specific Genes and their Expression in the Life History of the Red Alga Bostrychia moritziana (Ceramiales, Rhodomelaceae).

Diverse sex determination mechanisms have been reported in eukaryotes, but little is known about the genetic pathways leading to sex determination in red algae. Sex-specific genes that could be involved in sex determination and sexual differentiation were investigated in the red alga Bostrychia moritziana by analyzing the transcriptomes of various phases including males, females, and tetrasporophytes. Sex dominantly expressed genes which showed >10-fold difference between sexes was isolated using comparative RNA-seq analysis. We found 19 gene homologues, 10 from males, and nine from females, that were found only in one sex in genomic amplification using strains collected from five different localities. Most of the sex-specific genes are involved in important cellular processes including chromosome segregation, nucleo-cytoplasmic protein shuttling, or tRNA modification. Quantitative PCR analysis showed that some sex-specific genes were differently regulated during critical events of sexual reproduction like fertilization and carposporophyte development. We could localize the expression of a male-specific gene in spermatia before and after gamete binding using RNA insitu hybridization. Amino acid sequence identity between male and female homologues of importin alpha gene and PreQ(0) reductase were highly divergent (75% and 74%, respectively), suggesting that these divergent homologues are on non-recombining UV-type chromosomes in their respective sexes. Another set of transcripts were found that were sex dominantly expressed, but not sex-specific. Nineteen out of 39 sex dominantly expressed transcripts were annotated to transposable elements. Our results suggest that sexual differentiation in B.moritziana may be achieved by multi-level regulation of cellular processes, both from genes present only in one sex and differential expression of shared genes.

Comparative transcriptome analysis provides insight into the molecular mechanisms of anther dehiscence in eggplant (Solanum melongena L.)

Anther dehiscence releases pollen and therefore is a key event in plant sexual reproduction. Although anther dehiscence has been intensively studied in some plants, such as Arabidopsis thaliana and rice (Oryza sativa), the molecular mechanism of anther dehiscence in eggplant (Solanum melongena) is largely unknown. To provide insight into this mechanism, we used RNA-sequencing (RNA-seq) to analyze the transcriptomic profiles of one natural male-fertile line (F142) and two male-sterile lines (S12 and S13). We assembled 88,414 unigenes and identified 3446 differentially expressed genes (DEGs). GO and KEGG analysis indicated that these DEGs were mainly involved in “metabolic process”, “catalytic activity”, “biosynthesis of amino acids”, and “carbon metabolism”. The present study provides comprehensive transcriptomic profiles of eggplants that do and do not undergo anther dehiscence, and identifies a number of genes and pathways associated with anther dehiscence. The information deepens our understanding of the molecular mechanisms of anther dehiscence in eggplant.

The Biological Function of Extracellular Vesicles during Fertilization, Early Embryo-Maternal Crosstalk and Their Involvement in Reproduction: Review and Overview.

Secretory extracellular vesicles (EVs) are membrane-enclosed microparticles that mediate cell to cell communication in proximity to, or distant from, the cell of origin. Cells release a heterogeneous spectrum of EVs depending on their physiologic and metabolic state. Extracellular vesicles are generally classified as either exosomes or microvesicles depending on their size and biogenesis. Extracellular vesicles mediate temporal and spatial interaction during many events in sexual reproduction and supporting embryo-maternal dialogue. Although many omic technologies provide detailed understanding of the molecular cargo of EVs, the difficulty in obtaining populations of homogeneous EVs makes difficult to interpret the molecular profile of the molecules derived from a miscellaneous EV population. Notwithstanding, molecular characterization of EVs isolated in physiological and pathological conditions may increase our understanding of reproductive and obstetric diseases and assist the search for potential non-invasive biomarkers. Moreover, a more precise vision of the cocktail of biomolecules inside the EVs mediating communication between the embryo and mother could provide new insights to optimize the therapeutic action and safety of EV use.

Dynamics of an intense Alexandrium catenella red tide in the Gulf of Maine: satellite observations and numerical modeling

In July 2009, an unusually intense bloom of the toxic dinoflagellate Alexandrium catenella occurred in the Gulf of Maine. The bloom reached high concentrations (from hundreds of thousands to one million cells L−1) that discolored the water and exceeded normal bloom concentrations by a factor of 1000. Using Medium Resolution Imaging Spectrometer (MERIS) imagery processed to target chlorophyll concentrations (>2 µg L−1), patches of intense A. catenella concentration were identified that were consistent with the highly localized cell concentrations observed from ship surveys. The bloom patches were generally aligned with the edge of coastal waters with high-absorption. Dense bloom patches moved onshore in response to a downwelling event, persisted for approximately one week, then dispersed rapidly over a few days and did not reappear. Coupled physical-biological model simulations showed that wind forcing was an important factor in transporting cells onshore. Upward swimming behavior facilitated the horizontal cell aggregation, increasing the simulated maximum depth-integrated cell concentration by up to a factor of 40. Vertical convergence of cells, due to active swimming of A. catenella from the subsurface to the top layer, could explain the additional 25-fold intensification (25 × 40=1000-fold) needed to reach the bloom concentrations that discolored the water. A model simulation that considered upward swimming overestimated cell concentrations downstream of the intense aggregation. This discrepancy between model and observed concentrations suggested a loss of cells from the water column at a time that corresponded to the start of encystment. These results indicated that the joint effect of upward swimming, horizontal convergence, and wind-driven flow contributed to the red water event, which might have promoted the sexual reproduction event that preceded the encystment process.

Mating type specific transcriptomic response to sex inducing pheromone in the pennate diatom Seminavis robusta

Sexual reproduction is a fundamental phase in the life cycle of most diatoms. Despite its role as a source of genetic variation, it is rarely reported in natural circumstances and its molecular foundations remain largely unknown. Here, we integrate independent transcriptomic datasets to prioritize genes responding to sex inducing pheromones (SIPs) in the pennate diatom Seminavis robusta. We observe marked gene expression changes associated with SIP treatment in both mating types, including an inhibition of S phase progression, chloroplast division, mitosis, and cell wall formation. Meanwhile, meiotic genes are upregulated in response to SIP, including a sexually induced diatom specific cyclin. Our data further suggest an important role for reactive oxygen species, energy metabolism, and cGMP signaling during the early stages of sexual reproduction. In addition, we identify several genes with a mating type specific response to SIP, and link their expression pattern with physiological specialization, such as the production of the attraction pheromone diproline in mating type − (MT−) and mate-searching behavior in mating type + (MT+). Combined, our results provide a model for early sexual reproduction in pennate diatoms and significantly expand the suite of target genes to detect sexual reproduction events in natural diatom populations.

Boosting autophagy in sexual reproduction: a plant perspective.

The key process of sexual reproduction is the successful fusion of the sperm and egg cell. Distinct from dynamic and flagellated animal sperm cells, higher flowering plant sperm cells are immotile. Therefore, plants have evolved a novel reproductive system to achieve fertilization and generate progenies. Plant sexual reproduction consists of multiple steps, mainly including gametophyte development, pollen-pistil recognition, pollen germination, double fertilization and postfertilization. During reproduction, active production, consumption and recycling of cellular components and energy are critically required to achieve fertilization. However, the underlying machinery of cellular degradation and turnover remains largely unexplored. Autophagy, the major catabolic pathway in eukaryotic cells, participates in regulating multiple aspects of plant activities, including abiotic and biotic stress resistance, pathogen response, senescence, nutrient remobilization and plant development. Nevertheless, a key unanswered question is how autophagy regulates plant fertilization and reproduction. Here, we focus on comparing and contrasting autophagy in several key reproductive processes of plant and animal systems to feature important distinctions and highlight future research directions of autophagy in angiosperm reproduction. We further discuss the potential crosstalk between autophagy and programmed cell death, which are often considered as two disconnected events in plant sexual reproduction.

Sexual reproduction vs. clonal propagation in the recovery of a seagrass meadow after an extreme weather event

Marine flowering plants can reproduce sexually and clonally, and the relative contribution of these two modes can be dependent on the environmental conditions. Zostera marina, a seagrass widely distributed in the northern hemisphere, can form annual and perennial meadows with different proportions of sexual versus clonal propagation depending on the environmental disturbance regime. We study the hypothesis that the contribution of sexual propagation varies during the recovery of a seagrass meadow. In this case study, we compare the proportion of sexual versus clonal propagation of a perennial Z. marina meadow before its disappearance due to winter storms and after recovery. Before disturbance, genotypic diversity was high, indicating frequent sexual reproduction events likely to create an abundant seed bank. Seedling germination allowed the population to recover after the extreme disturbance. As months passed, seedlings became rare and finally absent, giving place to adult shoots. In an advanced stage of colonization, the shoots colonized the area by vegetative growth, which lowered the genotypic diversity. Despite this reduction over time, the genotypic diversity of the new meadow is still high, demonstrating the importance of sexual reproduction in meadow recovery and persistence.

Temporal changes in size distributions of the Southern Ocean diatom Fragilariopsis kerguelensis through high-throughput microscopy of sediment trap samples

Some aspects of the life cycle of the Southern Ocean diatom Fragilariopsis kerguelensis have been investigated previously, but many of its details have not been surveyed in nature. We investigated material from a two-year sediment trap time series by high-throughput imaging and image analysis, looking for morphometric signals of life cycle stages. Valve length distributions appeared close to unimodal but positively (right-) skewed. Size cohorts resulting from synchronized sexual reproduction events were not clearly distinguishable. Nevertheless, based on changes in valve length distributions, we found three general seasonal phases. These corresponded to periods of proliferation (with higher proportions of smaller cells during late spring/early summer), cessation of growth (relative loss of smaller cells during late summer/early autumn), and overwintering (little change in size distributions, with an increased proportion of large cells). We discuss possible causes of these signals, and their relevance to growth, sexual activity and adaption to environmental conditions, such as grazing pressures and the need for an overwintering strategy.

Novel insights into the genetic diversity and clonal structure of natural trembling aspen (Populus tremuloides Michx.) populations: A transcontinental study

AbstractAimDistribution‐wide trends in climate variability significantly influence the genetic diversity, differentiation and population structure of tree species. This study investigates the effects of disturbances such as fire, fragmentation and climate on modern‐day genetic patterns and clonal structures of trembling aspen at the transcontinental scale.LocationNorth American boreal zone.TaxonTrembling aspen (Populus tremuloides Michx.).MethodsOne thousand two hundred individuals in a 30‐site network (40 trees per location) were genotyped with neutral genetic markers and studied in relation to regional differences in climate and surrounding site conditions (aridity, fire cycle, fragmentation). Multiple linear regression models and variance analysis were used to test relationships between genetic indices, structural parameters and the surrounding site factors.ResultsOverall, a high percentage of single ramet clones (SRC) and clonal diversity was detected and assumed to be the consequence of multiple sexual reproduction events that took place at all sites, together with suckering, which shapes the clonal structure of populations. Neutral genetic diversity and clonal structure suggested no substantial differences among sites, which were categorized into climate moisture index (CMI) classes; aspen stands across Canada were highly similar from a genetic point of view. Allelic richness (AR) and the average number of alleles (Na) varied significantly among clonal organization groups, and landscape fragmentation and a higher frequency of fires showed a negative influence on the levels of genetic diversity.Main conclusionsOur results are inconsistent with the idea that the genotypic diversity of trembling aspen is related to the intensity of disturbance within the boreal forest. It appears that species‐specific disturbance responses and post‐fire recruitment mechanisms are more important than dominant ecological factors, such as climate and fire regimes, in shaping distribution‐wide patterns of neutral genetic variation and clonal structure.

AFLP markers show low levels of clonal propagation and high genotypic diversity in the rare, southernmost populations of Linnaea borealis L. (Caprifoliaceae) in the Western Alps.

In plants, clonal propagation is a common reproductive strategy in parallel to sexual reproduction. It has both advantages and drawbacks, and the potential complete loss of sexual reproduction causes serious conservation concerns, especially because population maintenance then only relies on adult survival and low genetic diversity leads to decreased adaptive potential. We investigated the rare, southernmost populations of the mostly circumboreal twinflower Linnaea borealis, located in the Western Alps. Based on 105 AFLP markers and 118 leaf samples, including replicates, we estimated the genetic similarity threshold above which samples belong to a single clone. Although the species is known for extensive clonal propagation, we observed high genotypic diversity within the seven studied populations and almost all samples were genetically distinct. Nevertheless, some clonal samples were detected in two populations, separated by up to 180m. We found a strong genetic differentiation among populations (overall Fst = 0.38), which was congruent with the previously documented high plastid diversity in the region. We therefore hypothesize that Alpine populations are relicts of the Quaternary glacial periods, when the species probably survived at these lower latitudes before colonizing Northern Europe. Regarding conservation, our results suggest that most extant plants result from sexual reproduction and that populations are not highly threatened. Nevertheless, since clones can be very long-lived and almost no seedlings were observed in recent years, events of sexual reproduction may be ancient. The current reproductive dynamics should therefore be studied to estimate e.g. pollinators activity, proportions of flowering plants, and seed set.