The combination of a flow cytometric seed screen and genotyping of each single seed offers a cost-effective approach to detecting complex reproductive pathways in flowering plants. Reproduction may be seen as one of the driving forces of evolution. Flow cytometric seed screen and genotyping of parents and progeny are commonly employed techniques to discern various modes of reproduction in flowering plants. Nevertheless, both methods possess limitations constraining their individual capacity to investigate reproductive modes thoroughly. We implemented both methods in a novel manner to analyse reproduction pathways using a carefully selected material of parental individuals and their seed progeny. The significant advantage of this approach lies in its ability to apply both methods to a single seed. The introduced methodology provides valuable insights into discerning the levels of apomixis, sexuality, and selfing in complex Rubus taxa. The results may be explained by the occurrence of automixis in Rubus, which warrants further investigation. The approach showcased its effectiveness in a different apomictic system, specifically in Taraxacum. Our study presents a comprehensive methodological approach for determining the mode of reproduction where flow cytometry loses its potential. It provides a reliable and cost-effective method with significant potential in biosystematics, population genetics, and crop breeding.

• Keywords
• Rubus, Taraxacum, Apomixis, Automixis, FCSS, SSR-seq,
• MeSH
• Apomixis * genetics   physiology   MeSH
• Genotype MeSH
• Flow Cytometry * MeSH
• Reproduction MeSH
• Seeds * genetics   physiology   MeSH
• Taraxacum physiology   genetics   MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH

BACKGROUND AND AIMS: Rubus ser. Glandulosi provides a unique model of geographical parthenogenesis on a homoploid (2n = 4x) level. We aim to characterize evolutionary and phylogeographical patterns in this taxon and shed light on the geographical differentiation of apomicts and sexuals. Ultimately, we aim to evaluate the importance of phylogeography in the formation of geographical parthenogenesis. METHODS: Rubus ser. Glandulosi was sampled across its Eurasian range together with other co-occurring Rubus taxa (587 individuals in total). Double-digest restriction site-associated DNA sequencing (ddRADseq) and modelling of suitable climate were used for evolutionary inferences. KEY RESULTS: Six ancestral species were identified that contributed to the contemporary gene pool of R. ser. Glandulosi. Sexuals were introgressed from Rubus dolichocarpus and Rubus moschus in West Asia and from Rubus ulmifolius agg., Rubus canescens and Rubus incanescens in Europe, whereas apomicts were characterized by alleles of Rubus subsect. Rubus. Gene flow between sexuals and apomicts was also detected, as was occasional hybridization with other taxa. CONCLUSIONS: We hypothesize that sexuals survived the last glacial period in several large southern refugia, whereas apomicts were mostly restricted to southern France, whence they quickly recolonized Central and Western Europe. The secondary contact of sexuals and apomicts was probably the principal factor that established geographical parthenogenesis in R. ser. Glandulosi. Sexual populations are not impoverished in genetic diversity along their borderline with apomicts, and maladaptive population genetic processes probably did not shape the geographical patterns.

• Keywords
• Rubus subgen. Rubus, Apomixis, ddRADseq, geographical parthenogenesis, introgression, private alleles,
• MeSH
• Apomixis genetics   MeSH
• Biological Evolution MeSH
• Phylogeny MeSH
• Phylogeography * MeSH
• Genetic Variation MeSH
• Parthenogenesis genetics   MeSH
• Rosaceae * genetics   physiology   MeSH
• Gene Flow MeSH
• Publication type
• Journal Article MeSH
• Geographicals
• Asia MeSH
• Europe MeSH

PREMISE: Apomixis in ferns is relatively common and obligatory. Sterile hybrids may restore fertility via apomixis at a cost of long-term genetic stagnation. In this study, we outlined apomixis as a possible temporary phase leading to sexuality and analyzed factors relating to transitioning to and away from apomixis, such as unreduced and reduced spore formation in apomict and apo-sex hybrid ferns. METHODS: We analyzed the genome size of 15 fern species or hybrids ("taxa") via flow cytometry. The number of reduced and unreduced gametophytes was established as a proxy for viable spore formation of either type. We also calculated the spore abortion ratio (sign of reduced spores) in several taxa, including the apo-sex hybrid Dryopteris × critica and its 16 apomictically formed offspring. RESULTS: Four of 15 sampled taxa yielded offspring variable in genome size. Specifically, each variable taxon formed one viable reduced plant among 12-451 sampled gametophytes per taxon. Thus, haploid spore formation in the studied apomicts was very rare but possible. Spore abortion analyses indicated gradually decreasing abortion (haploid spore formation) over time. In Dryopteris × critica, abortion decreased from 93.8% to mean 89.5% in one generation. CONCLUSIONS: Our results support apomixis as a transitionary phase toward sexuality. Newly formed apomicts hybridize with sexual relatives and continue to form haploid spores early on. Thus, they may get the genomic content necessary for regular meiosis and restore sexuality. If the missing relative goes extinct, the lineage gets locked into apomixis as may be the case with the Dryopteris affinis complex.

• Keywords
• Dryopteris, apogamy, apomixis, evolutionary biology, fern life cycle, flow cytometry, gametophyte, genome size, pteridophytes, reproduction,
• MeSH
• Apomixis * genetics   MeSH
• Genome Size * MeSH
• Genome, Plant * MeSH
• Hybridization, Genetic MeSH
• Ferns * genetics   physiology   MeSH
• Spores * physiology   genetics   MeSH
• Publication type
• Journal Article MeSH

Asexual organisms often differ in their geographic distributions from their sexual relatives. This phenomenon, termed geographic parthenogenesis, has long been known, but the underlying factors behind its diverse patterns have been under dispute. Particularly problematic is an association between asexuality and polyploidy in most taxa. Here, we present a new system of geographic parthenogenesis on the tetraploid level, promising new insights into this complex topic. We used flow cytometric seed screen and microsatellite genotyping to characterise the patterns of distribution of sexuals and apomicts and genotypic distributions in Rubus ser. Glandulosi across its range. Ecological modelling and local-scale vegetation and soil analyses were used to test for niche differentiation between the reproductive groups. Apomicts were detected only in North-western Europe, sexuals in the rest of the range in Europe and West Asia, with a sharp borderline stretched across Central Europe. Despite that, we found no significant differences in ecological niches. Genotypic richness distributions suggested independence of the reproductive groups and a secondary contact. We argue that unless a niche differentiation (resulting from polyploidy and/or hybridity) evolves, the main factors behind the patterns of geographic parthenogenesis in plants are phylogeographic history and neutral microevolutionary processes, such as clonal turnover.

• Keywords
• Rubus ser. Glandulosi, apomixis, clonal turnover, genotypic diversity, geographical parthenogenesis, niche differentiation,
• MeSH
• Apomixis * MeSH
• Parthenogenesis genetics   MeSH
• Ploidies MeSH
• Polyploidy MeSH
• Rubus * MeSH
• Publication type
• Journal Article MeSH

In higher plants, sexual reproduction is characterized by meiosis of the first cells of the germlines, and double fertilization of the egg and central cell after gametogenesis. In contrast, in apomicts of the genus Boechera, meiosis is omitted or altered and only the central cell requires fertilization, while the embryo forms parthenogenetically from the egg cell. To deepen the understanding of the transcriptional basis underlying these differences, we applied RNA-seq to compare expression in reproductive tissues of different Boechera accessions. This confirmed previous evidence of an enrichment of RNA helicases in plant germlines. Furthermore, few RNA helicases were differentially expressed in female reproductive ovule tissues harboring mature gametophytes from apomictic and sexual accessions. For some of these genes, we further found evidence for a complex recent evolutionary history. This included a homolog of Arabidopsis thaliana FASCIATED STEM4 (FAS4). In contrast to AtFAS4, which is a single-copy gene, FAS4 is represented by three homologs in Boechera, suggesting a potential for subfunctionalization to modulate reproductive development. To gain first insights into functional roles of FAS4, we studied Arabidopsis lines carrying mutant alleles. This identified the crucial importance of AtFAS4 for reproduction, as we observed developmental defects and arrest during male and female gametogenesis.

• Keywords
• Boechera, FASCIATED STEM4, Apomixis, RNA helicase, evolution, gametogenesis, reproduction, transcriptome,
• MeSH
• Apomixis * genetics   MeSH
• Arabidopsis * genetics   MeSH
• Biological Evolution MeSH
• Brassicaceae * genetics   MeSH
• Cell Cycle MeSH
• Reproduction genetics   MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH

PREMISE: Apomixis and hybridization are two essential and complementary factors in the evolution of plants, including ferns. Hybridization combines characteristics from different species, while apomixis conserves features within a lineage. When combined, these two processes result in apo-sex hybrids. The conditions leading to the formation of these hybrids are poorly understood in ferns. METHODS: We cultivated spores from 66 fern samples (43 apomicts, 7 apo-sex hybrids, and 16 sexuals), and measured their development in vitro over 16 weeks. We evaluated germination, lateral meristem formation rates, sexual expression, and production of sporophytes and then compared ontogenetic patterns among the three groups. RESULTS: The three examined groups formed antheridia (male gametangia) but differed in overall gametophyte development. Sexual species created archegonia (female, 86% of viable samples), but no sporophytes. Apomicts rarely created nonfunctional archegonia (8%) but usually produced apogamous sporophytes (75%). Surprisingly, apomictic and sexual species showed similar development speed. The sexually reproducing parents of viable studied hybrids formed about twice as many meristic gametophytes as the apomictic parents (39% vs. 20%, respectively). CONCLUSIONS: We present the most thorough comparison of gametangial development of sexual and apomictic ferns, to date. Despite expectations, apomictic reproduction might not lead to earlier sporophyte formation. Apomicts produce functional sperm and thus can contribute this type of gamete to their hybrids. The development patterns found in the parents of hybrids indicate a possible increase of hybridization rates by antheridiogens. The apo-sex hybrids always inherit the apomictic reproductive strategy and are thus capable of self-perpetuation.

• Keywords
• Dryopteris, agamospory, antheridia, apo-sex hybrid, apogamy, archegonia, gametes, hybridization, pteridophytes, wood fern,
• MeSH
• Apomixis * genetics   MeSH
• Ferns * genetics   MeSH
• Humans MeSH
• Fathers MeSH
• Reproduction MeSH
• Germ Cells, Plant MeSH
• Check Tag
• Humans MeSH
• Male MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH

High reproductive assurance is regarded as a key advantage of uniparentally reproducing organisms for establishing a new population. This demographic benefit should especially be relevant for plants with autonomous apomixis, that is those which produce seeds completely independently from mates and pollinators. Indeed, many autonomous apomicts occupy larger distributional ranges when compared to their sexual relatives, showing geographical parthenogenesis patterns. However, uniparental reproduction advantage has only rarely been quantified in natural populations and results provided a mixed support, partly because allopatric sexual and asexual populations were exposed to different environmental and pollination conditions causing considerable between-population variation in the level of reproductive assurance. Here, we compared the level and stability of reproductive assurance between sexual self-incompatible and asexual autonomously apomictic plants of Hieracium alpinum (Asteraceae) cultivated in a sympatric low-density population with two levels of spatial clumping of sexual plants. Overall, we found that the realized seed set (i.e. proportion of well-developed seeds per capitulum) of asexuals was ca. 3 times greater than that of sexuals (83% vs. 27%), whereas the variance of this trait expressed as coefficient of variation was ca. 4 times smaller in asexuals compared with sexuals (19% vs. 83%). Solitary sexual plants had more than 2 times lower realized seed set when compared to clumps composed of two spatially close (20-30 cm) sexual plants (13% vs. 34%). Our study provides experimental evidence for benefit of uniparental reproduction of asexuals in a sympatric situation when the availability of mates is limited. This, together with unpredictability of pollinator environment could provide autonomous apomicts with an ultimate demographic superiority during colonization reflected in geographical parthenogenesis observed in this species.

• Keywords
• Baker law, apomixis, colonization, geographical parthenogenesis, long-distance dispersal, mate limitation, pollen limitation, polyploidy, seed set,
• MeSH
• Apomixis * genetics   MeSH
• Pollen MeSH
• Plants MeSH
• Reproduction MeSH
• Seeds MeSH
• Sympatry MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH

Sex expression of homosporous ferns is controlled by multiple factors, one being the antheridiogen system. Antheridiogens are pheromones released by sexually mature female fern gametophytes, turning nearby asexual gametophytes precociously male. Nevertheless, not all species respond. It is still unknown how many fern species use antheridiogens, how the antheridiogen system evolved, and whether it is affected by polyploidy and/or apomixis. We tested the response of 68 fern species to antheridiogens in cultivation. These results were combined with a comprehensive review of literature to form the largest dataset of antheridiogen interactions to date. Analyzed species also were coded as apomictic or sexual and diploid or polyploid. Our final dataset contains a total of 498 interactions involving 208 species (c. 2% of all ferns). About 65% of studied species respond to antheridiogen. Multiple antheridiogen types were delimited and their evolution is discussed. Antheridiogen responsiveness was not significantly affected by apomixis or polyploidy. Antheridiogens are widely used by ferns to direct sex expression. The antheridiogen system likely evolved multiple times and provides homosporous ferns with the benefits often associated with heterospory, such as increased rates of outcrossing. Despite expectations, antheridiogens may be beneficial to polyploids and apomicts.

• Keywords
• antheridiogen, apomixis, ferns, gametophyte, germination, mating, polyploidy, sex expression,
• MeSH
• Apomixis * genetics   MeSH
• Diploidy MeSH
• Ferns * genetics   MeSH
• Polyploidy MeSH
• Germ Cells, Plant MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Review MeSH
• Research Support, U.S. Gov't, Non-P.H.S. MeSH

Gametophytic apomixis is a way of asexual plant reproduction by seeds. It should be advantageous under stressful high altitude or latitude environment where short growing seasons, low temperatures, low pollinator activity or unstable weather may hamper sexual reproduction. However, this hypothesis remains largely untested. Here, we assess the reproductive mode in 257 species belonging to 45 families from the world's broadest alpine belt (2800-6150 m) in NW Himalayas using flow cytometric seed screen. We found only 12 apomictic species, including several members of Poaceae (Festuca, Poa and Stipa), Rosaceae (Potentilla) and Ranunculaceae (Halerpestes, Ranunculus), which are families typical for high apomict frequency. However, several apomictic species were newly discovered, including the first known apomictic species from the family Biebersteiniaceae (Biebersteinia odora), and first apomicts from the genera Stipa (Stipa splendens) and Halerpestes (Halerpestes lancifolia). Apomicts showed no preference for higher elevations, even in these extreme Himalayan alpine habitats. Additional trait-based analyses revealed that apomicts differed from sexuals in comprising more rhizomatous graminoids and forbs, higher soil moisture demands, sharing the syndrome of dominant species with broad geographical and elevation ranges typical for the late-successional habitats. Apomicts differ from non-apomicts in greater ability of clonal propagation and preference for wetter, more productive habitats.

• MeSH
• Apomixis * MeSH
• Ecosystem * MeSH
• Phylogeny MeSH
• Plant Physiological Phenomena * MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH

Although reproductive assurance has been suggested to be one of the most important factors shaping the differential distributional patterns between sexuals and asexuals (geographic parthenogenesis), it has only rarely been studied in natural populations of vascular plants with autonomous apomixis. Moreover, there are almost no data concerning the putative relationship between the level of apomictic versus sexual plant reproduction on one hand, and reproductive assurance on the other. We assessed the level of sexual versus apomictic reproduction in diploid and triploid plants of Hieracium alpinum across its distributional range using flow cytometric analyses of seeds, and compared the level of potential and realized seed set, i.e. reproductive assurance, between the two cytotypes under field and greenhouse conditions. Flow cytometric screening of embryos and endosperms of more than 4,100 seeds showed that diploids produced solely diploid progeny sexually, while triploids produced triploid progeny by obligate apomixis. Potential fruit set was much the same in diploids and triploids from the field and the greenhouse experiment. While in the pollination-limited environment in the greenhouse apomictic triploids had considerably higher realized fruit set than sexual diploids, there was no significant difference between cytotypes under natural conditions. In addition, sexuals varied to a significantly larger extent in realized fruit set than asexuals under both natural and greenhouse conditions. Our results indicate that triploid plants reproduce by obligate apomixis, assuring more stable and predictable fruit reproduction when compared to sexual diploids. This advantage could provide apomictic triploids with a superior colonisation ability, mirrored in a strong geographic parthenogenesis pattern observed in this species.

• Keywords
• FCSS, Apomixis, breeding system, flow cytometry, fruit set, geographic parthenogenesis, polyploidy, reproductive assurance, unreduced gametes,
• MeSH
• Apomixis * MeSH
• Asteraceae genetics   physiology   MeSH
• Diploidy MeSH
• Endosperm physiology   MeSH
• Fruit growth & development   MeSH
• Flow Cytometry MeSH
• Reproduction MeSH
• Seeds growth & development   MeSH
• Triploidy * MeSH
• Publication type
• Journal Article MeSH