Apomictic Species Research Articles

Extinct or Not? Confirming the “Extinct” Status of Hieracium tolstoii (Asteraceae) with Integrated Taxonomic Investigation

Hieracium is one of the largest flowering plant genera that has recently experienced a rapid evolutionary radiation. Due to the massive morphological variation among species, frequent hybridization, and polyploidization events, combined with apomictic reproduction in polyploids, the concept of species in Hieracium is widely debated, and species identification is incredibly challenging. Taxonomic investigation can lead to changes in conservation status or, in some cases, to de-extinctions of species. Taxa with doubtful classification are particularly sensitive to this kind of problem. Hieracium tolstoii was a narrow Italian endemic species that is nowadays considered extinct. Described as a morphological intermediate form between two other species, no further investigations were conducted after its first description. To clarify H. tolstoii’s origin and ensure the validity of its current conservation status, we conducted a taxonomic investigation on herbaria specimens of the early XX century. Specimens were studied using a morphometric analysis that compared H. tolstoii with other closely related taxa. Moreover, we performed phylogenetic investigations using three plastid intergenic spacers to evaluate the relationship between species. Plastid markers revealed the presence of indels and SNPs in H. tolstoii sequences that differ from sequences of the supposed parental species. Morphometry revealed differences among species that led us to confirm the validity of H. tolstoii as an independent apomictic species and, therefore, unfortunately, its extinction.

Investigating sexual and asexual modes of reproduction in Palmer Amaranth (Amaranthus palmeri)

Abstract Palmer amaranth (Amaranthus palmeri S. Watson), a dioecious wind-pollinated plant, is one of the most troublesome crop weeds in the United States and is spreading northwards. The prodigious production of seed contributes to establishment of populations and spread across the landscape. Sexual reproduction via outcrossing is likely the primary mode of seed production for this dioecious plant. However, A. palmeri may also be capable of autonomous asexual seed production (apomixis), which could be beneficial during colonization. We conducted two studies of female isolation from pollen to investigate the propensity for autonomous seed production in 19 populations across eastern North America. In the first we observed low frequency seed production on many isolated females. Using flow cytometry of seed samples (FCSS) we primarily found patterns of ploidy consistent with sexual reproduction; no significant differences in ploidy between seeds produced on isolated females (putative apomicts) and non-isolated females (putatively sexual) were detected. We also investigated patterns of DNA content and found no evidence for polyploidy in 153 samples, which is often observed in apomictic species. The second female isolation trial utilized sex-specific molecular markers to identify and remove males prior to flowering, and we observed zero seed production. Overall, we did not detect evidence in support of apomixis in these populations of A. palmeri, suggesting that apomixis is unlikely to have played a role in the northward advance of this species in eastern North America. We also investigated whether there is variation between females and males in size and secondary reproductive traits. We found evidence for sexual dimorphism in three of six traits investigated: females are taller at senescence, produce longer secondary branches, and more axillary flowers than males. Differences in cost of reproduction and strategies for pollen release vs. pollen capture are likely factors shaping the evolution of sexual dimorphism in this wind-pollinated dioecious plant.

Taraxacum erythroboreale (Asteraceae) — новый вид предположительно межсекционного гибридного происхождения с широким распространением на севере Европейской России (Taraxacum Notulae Rossiae Boreali Europeae II)

Taraxacum erythroboreale Efimov, a new apomictic species, is described and illustrated. It occurs in Russia (North of the European part), in the Murmansk and Vologda regions, in the Komi Republic, being locally very abundant. It occupies mainly dry sandy roadsides and other similar ruderal places, and seems benefiting from the anthropogenic disturbance of the land. The new species may belong either to the section Erythrosperma (H. Lindb.) Dahlst., or have originated from the hybridization between this section and some species of Asiatic sections Stenoloba Kirschner et Štěpánek or Dissecta (Soest) R. Doll. The new species displays rather wide morphological polymorphism as to the presence/absence of purplish achene pigmentation, shape of outer phyllaries, corniculation of phyllaries, and in some other traits.

Apomixis goes a long way: Genetic evidence of persistence and long‐distance seed dispersal in an ancient landscape

AbstractAimApomixis is a widespread trait in extreme environments worldwide, yet phylogeographical studies for species exhibiting these complex reproductive systems are still limited to temperate zones in the Northern Hemisphere. Through analyses of a combination of adult plants and seedlings, and nuclear and chloroplast DNA, we assessed the contemporary genetic outcomes of apomixis and phylogeographical patterns in an arid unglaciated landscape to understand the evolutionary trajectory of apomictic species.LocationPilbara bioregion, north‐western Australia.TaxonSenna glutinosa subsp. glutinosa.Materials and MethodsSpatial patterns of genetic diversity and differentiation in three chloroplast regions and 17 nuclear microsatellite markers were evaluated in 480 plants from 20 populations of S. glutinosa subsp. glutinosa across the Pilbara bioregion. We also germinated and genotyped seed for a progeny analysis to quantify polyembryony and the relative frequency of asexual versus sexual reproduction.ResultsWe found frequent polyembryony and a high proportion of asexual offspring (84.56%), resulting in low within‐population genotypic diversity. The nuclear dataset identified 72 multilocus lineages (MLLs) with moderate pairwise population genetic fixation and low differentiation among populations with no geographically structured genetic clustering. Chloroplast DNA showed high haplotype diversity and a widespread distribution of haplotypes. Only one peripheral population presented a single unique haplotype and MLL.Main ConclusionsAnalysis of this woody perennial in an arid landscape shows the influence of apomixis on genetic patterns and species persistence. Haplotype diversity was indicative of long‐term persistence within the ancient Pilbara region. Widespread distribution of haplotypes and sharing of several nuclear MLLs among distant populations indicated extensive seed dispersal throughout the study area. The combination of polyploidy, facultative apomixis and extensive seed dispersal maintains genetic variability within local populations and promotes the spread of advantageous genotypes across wide geographical distances. Therefore, apomixis, rather than an evolutionary ‘dead‐end’, can be regarded as a mechanism facilitating evolutionary success of apomictic species in extreme and complex environments.

Immunolocalization of Pectins and Arabinogalactan Proteins in Young Ovules of Selected Amphimictic and Apomictic Species of the Asteraceae Family

Immunolocalization of Pectins and Arabinogalactan Proteins in Young Ovules of Selected Amphimictic and Apomictic Species of the Asteraceae Family

Apomixis occurs frequently along the entire American Cordillera

Abstract Apomictic plants mostly occur at higher latitudes and higher elevations, where they tend to occupy deglaciated areas. By analysing samples of the alpine floras of different latitudinal zones of the Americas (Argentina, Bolivia, Ecuador, USA), we investigated whether the proportion of gametophytic apomixis is comparable between the temperate alpine zones of the Northern Hemisphere and the Southern Hemisphere and whether the proportion of apomictic species increases from the alpine tropics towards the temperate alpine zone. The reproductive systems of 196 species and 275 individuals were determined by a flow-cytometric seed screening of 999 seeds. Phylogenetic logistic regression was used to evaluate differences in the proportion of apomictic species in samples of regional alpine floras. The apomictic embryo:endosperm ratio was determined for 33 species (two pseudogamous species and 31 species with autonomous apomixis) belonging to 28 genera of 13 families. The probability of a species being apomictic did not significantly differ between the regions. The highest probability of a species being apomictic was estimated for the northern temperate zone (29.6%), followed by the tropical zone (12.5%), and the southern temperate zone (11.9%). Our results show that asexual plant reproduction by apomixis is not restricted to the alpine zone of the Northern Hemisphere and its representation in alpine floras of the Southern Hemisphere, including the alpine tropics, has been substantially underestimated.

Twelve new species of Hieracium sect. Tridentata (Asteraceae) from southern Sweden

Twelve new, presumably apomictic species of Hieracium sect. Tridentata, namely H. arcanum, H. versifrons, H. hyperoptum, H. irrugantifrons, H. karolinae, H. leucophlebium, H. melanostigma, H. neomixopolium, H. oxyodontum, H. rostockense, H. subcreperum and H. verecundum are described from the Götaland region of southern Sweden. Apart from morphological descriptions, illustrations and maps showing the distribution of all the species are provided.

Hieracium lomniczkianum (Asteraceae), a new species in the H. wiesbaurianum aggregate from the Sudetes in Poland

Hieracium lomniczkianum is a new, apomictic species, discovered in the Karkonosze Mountains (Giant Mountains) in the Sudetes, SW Poland. It belongs to the H. wiesbaurianum aggregate which comprises taxa intermediate in characters between H. bifidum s.lat. and H. schmidtii s.lat. The new species is distinguished by a slender stem with small capitula, and by narrow, weakly dentate rosette leaves which are gradually tapered to a winged petiole.

Taxa of Vascular Plants Endemic to the Pannonicum Floristic Region

Following the work of Tatár (1939), no new revised and detailed list was made of endemic plants of the Pannonicum phytogeographical region, which takes into account the latest research results. A survey of vascular plants endemic and subendemic to the Pannonicum is presented here based on a critical revision of published and sometimes unpublished data on contemporary taxonomic and chorological knowledge. For this, it was necessary to review the delineation of Pannonicum and the problem of drawing the boundaries. I would also like to draw the attention to the Pannonian flora islands outside the Carpathians, which descend along the sandy alluvium of the Danube. The research covers 11 countries: Austria, Croatia, the Czech Republic, Hungary, Romania, Serbia, Slovakia, Slovenia and a small part of Bosnia and Herzegovina, Ukraine and Bulgaria (flora islands). The final evaluation of endemic status was made for 225 taxa of vascular plants, including 143 taxa confirmed as endemic or subendemic to the Pannonicum, 5 narrowly distributed taxa shared endemic of the Pannonicum and western part of the Carpaticum and 77 taxa are not endemic according to current taxonomic and phytogeographical knowledge (the list does not include hybrids). The final list of endemic and subendemic taxa includes 42 species, 29 subspecies and 73 apomictic species (including 47 taxa of Sorbus and 23 taxa of Taraxacum). Tatár mentions 55 taxa (without apomicts) of which only 29 (53%) are still considered endemic today. In terms of habitat preferences for (sub)endemic taxa most plants (excluding apomictic taxa) occur in rocky or sandy habitats.

Ovule Transcriptome Analysis Discloses Deregulation of Genes and Pathways in Sexual and Apomictic Limonium Species (Plumbaginaceae).

The genus Limonium Mill. (sea lavenders) includes species with sexual and apomixis reproductive strategies, although the genes involved in these processes are unknown. To explore the mechanisms beyond these reproduction modes, transcriptome profiling of sexual, male sterile, and facultative apomictic species was carried out using ovules from different developmental stages. In total, 15,166 unigenes were found to be differentially expressed with apomictic vs. sexual reproduction, of which 4275 were uniquely annotated using an Arabidopsis thaliana database, with different regulations according to each stage and/or species compared. Gene ontology (GO) enrichment analysis indicated that genes related to tubulin, actin, the ubiquitin degradation process, reactive oxygen species scavenging, hormone signaling such as the ethylene signaling pathway and gibberellic acid-dependent signal, and transcription factors were found among differentially expressed genes (DEGs) between apomictic and sexual plants. We found that 24% of uniquely annotated DEGs were likely to be implicated in flower development, male sterility, pollen formation, pollen-stigma interactions, and pollen tube formation. The present study identifies candidate genes that are highly associated with distinct reproductive modes and sheds light on the molecular mechanisms of apomixis expression in Limonium sp.

A new species in Hieracium sect. Alpina (Asteraceae) from the Eastern Carpathians in Romania

Hieracium ciucasense is a new, apomictic species described from the Ciucaş Mountains in the Eastern Carpathians, Romania. It belongs to the H. pietroszense aggregate comprising taxa of presumably hybrid origin between H. alpinum L. and H. bifidum s. lat. The new species is illustrated with photographs of the holotype and living plants; it differs from the other taxa of the H. pietroszense aggregate in the Carpathians in that the leaves are brown-purple spotted. The distribution of the H. pietroszense aggregate in the Carpathians is mapped.

Phylogeography of Eriotheca species complex: insights into the origin and range expansion of apomictic and polyploid trees in Neotropical Savannas.

Polyploidy and whole genome duplication are major evolutionary drivers in plants. Climate variations during the Pleistocene have influenced distribution and range expansion worldwide. Similar trends have been reported for Cerrado plants, but no attempt has been made to link phylogeography with ploidy and breeding changes. Thus, we aimed to (i) assess ploidy and genome size of Eriotheca estevesiae Carv.-Sobr., and compare it with E. pubescens (Mart.) Schott & Endl. (Both included into the Eriotheca Stellate Trichome Species Complex - ESTSC). (ii) Subsequently, we investigated their phylogeography to see whether genetic structure and range expansion trends were similar to those previously described for the Cerrado biome. Finally (iii), we discuss whether ESTSC phylogeographic patterns could be associated with geographic parthenogenesis processes. Common cytogenetic techniques and flow cytometry were used to confirm chromosome number and genome size of E. estevesiae. We used three cpDNA regions to analyse 14 ESTSC Cerrado populations, for which we also obtained ploidy level and breeding information. We investigated haplotype diversity, population structure and tested neutrality, aiming to reconstruct phylogeographic scenarios. We found three ploidy levels and eight cpDNA haplotypes in ESTSC, one shared by most populations. Haplotype and ploidy distribution corroborated that E. pubescens, the widely distributed polyploid and apomictic species, may have originated from northern diploid and probably sexual E. estevesiae. Matrilinear cpDNA links support the idea that apomixis and polyploidy in ESTSC may have allowed range expansion during the Pleistocene, in a process analogous to the geographic parthenogenesis described elsewhere.

Genome-wide family prediction unveils molecular mechanisms underlying the regulation of agronomic traits in Urochloa ruziziensis.

Tropical forage grasses, particularly those belonging to the Urochloa genus, play a crucial role in cattle production and serve as the main food source for animals in tropical and subtropical regions. The majority of these species are apomictic and tetraploid, highlighting the significance of U. ruziziensis, a sexual diploid species that can be tetraploidized for use in interspecific crosses with apomictic species. As a means to support breeding programs, our study investigates the feasibility of genome-wide family prediction in U. ruziziensis families to predict agronomic traits. Fifty half-sibling families were assessed for green matter yield, dry matter yield, regrowth capacity, leaf dry matter, and stem dry matter across different clippings established in contrasting seasons with varying available water capacity. Genotyping was performed using a genotyping-by-sequencing approach based on DNA samples from family pools. In addition to conventional genomic prediction methods, machine learning and feature selection algorithms were employed to reduce the necessary number of markers for prediction and enhance predictive accuracy across phenotypes. To explore the regulation of agronomic traits, our study evaluated the significance of selected markers for prediction using a tree-based approach, potentially linking these regions to quantitative trait loci (QTLs). In a multiomic approach, genes from the species transcriptome were mapped and correlated to those markers. A gene coexpression network was modeled with gene expression estimates from a diverse set of U. ruziziensis genotypes, enabling a comprehensive investigation of molecular mechanisms associated with these regions. The heritabilities of the evaluated traits ranged from 0.44 to 0.92. A total of 28,106 filtered SNPs were used to predict phenotypic measurements, achieving a mean predictive ability of 0.762. By employing feature selection techniques, we could reduce the dimensionality of SNP datasets, revealing potential genotype-phenotype associations. The functional annotation of genes near these markers revealed associations with auxin transport and biosynthesis of lignin, flavonol, and folic acid. Further exploration with the gene coexpression network uncovered associations with DNA metabolism, stress response, and circadian rhythm. These genes and regions represent important targets for expanding our understanding of the metabolic regulation of agronomic traits and offer valuable insights applicable to species breeding. Our work represents an innovative contribution to molecular breeding techniques for tropical forages, presenting a viable marker-assisted breeding approach and identifying target regions for future molecular studies on these agronomic traits.

ROWANS, WHITEBEAMS AND SERVICE TREES

ROWANS, WHITEBEAMS AND SERVICE TREES

Hybrids between Rubus caesius and Rubus sect. Corylifolii (Rosaceae) and their relation to R. cyclomorphus, R. tiliaster, R. glauciformis, R. slesvicensis and R. firmus

Using flow cytometry and microsatellite DNA analysis (MSDA), we studied hybrids between Rubus caesius and various other Rubus species, with an emphasis on Sweden. We show that hybrids between Rubus caesius and Rubus sect. Corylifolii arise easily. They show a large variation in morphology, but can normally be recognised by a number of characters. They are typically hexaploids, but ~13% of the hybrids are pentaploids and ~8% tetraploids. With MSDA, they are harder to identify than hybrids with R. idaeus, partly because all Corylifolii species have themselves arisen from R. caesius hybrids and therefore share many alleles with R. caesius, partly because hybridisation with R. caesius seems to give rise to some variation in microsatellite regions. Hybrids with species of R. sect. Rubus are much rarer in Sweden and we have only identified one such case. We show that R. cyclomorphus and R. tiliaster are not proper apomictic species, at least not in Sweden, but rather a collection of genetically unrelated hybrids between R. caesius and R. raduloides or R. camptostachys. MSDA analysis of R. slesvicensis and R. firmus shows that these species are problematic. We identify two distinct taxa, one from Skåne in Sweden, which we describe as a new species, R. ruboculus and another from the neighbourhood of Viborg and Schleswig, although the latter is probably not identical with R. slesvicensis s.s. All the other samples form a diverse group of putative R. caesius hybrids at three different ploidy levels. Rubus glauciformis is uniform in Småland, Öland and Blekinge, but becomes harder to distinguish from various hybrids in Skåne. We conclude that once R. caesius and R. idaeus hybrids are properly identified, along with a small number of new apomictic species (often with a local distribution), the genus Rubus does not pose any serious taxonomical problems in Sweden.

Sexual and reproductive systems of woody species in vereda are distributed according to the life form and habitat occurrence

AbstractIn Cerrado humid areas, veredas are considered as hygrophilous communities, with an herbaceous/shrub stratum in open areas and an arboreal stratum in forest areas. We identified the sexual and reproductive systems of woody species, and the distribution of these systems among the different habitats and plants habits in this ecosystem. We investigated whether the occurrence of sexual and reproductive systems may be related to ecological and/or evolutionary factors and if hermaphroditism predominates in open areas. In this case, we evaluated which reproductive strategies promote cross‐pollination and which species are dependent on pollinating agents to promote the reproductive process. We selected the species according to a phytosociological study, using plots distributed in parallel transects and allocated in the hygrophilous forest and open areas. Floral morphology analyses, self‐pollination treatments, presence of barriers that prevent self‐pollination, phylogenetic signal and literature survey were performed. We did not find phylogenetic signal evidence for the traits evaluated. Most species are trees, followed by shrubs and sub‐shrubs. Dioecy predominated among tree species in the open area, with hermaphroditism more related to shrub species in the open areas. Shrubs and sub‐shrubs were mainly represented by autogamous and apomictic species. Despite autogamy and apomixis were related to the open areas, we found no significant difference between the habitats. In veredas, that are considered a poorly resilient environment that has constant problems with recent anthropogenic activities, we expected a high number of autogamous and apomictic species. However, the results showed that most species have characteristics that promote allogamy and are pollinating agents dependent for reproduction.

Controlled Induction of Parthenogenesis in Transgenic Rice via Post-translational Activation of PsASGR-BBML.

Modern plant breeding programs rely heavily on the generation of homozygous lines, with the traditional process requiring the inbreeding of a heterozygous cross for five to six generations. Doubled haploid (DH) technology, a process of generating haploid plants from an initial heterozygote, followed by chromosome doubling, reduces the process to two generations. Currently established in vitro methods of haploid induction include androgenesis and gynogenesis, while in vivo methods are based on uni-parental genome elimination. Parthenogenesis, embryogenesis from unfertilized egg cells, presents another potential method of haploid induction. PsASGR-BABY BOOM-like, an AP2 transcription factor, induces parthenogenesis in a natural apomictic species, Pennisetum squamulatum (Cenchrus squamulatus) and PsASGR-BBML transgenes promote parthenogenesis in several crop plants, including rice, maize, and pearl millet. The dominant nature of PsASGR-BBML transgenes impedes their use in DH technology. Using a glucocorticoid-based post-translational regulation system and watering with a 100 μM DEX solution before anthesis, PsASGR-BBML can be regulated at the flowering stage to promote parthenogenesis. Conditional expression presents a novel opportunity to use parthenogenetic genes in DH production technology and to elucidate the molecular mechanism underlying parthenogenetic embryogenesis.

Comparative Expression Profiles of SUVH7 in Sexual and Apomict Boechera spp. Display Differential Expression

Genomic imprinting is parent-of-origin specific gene expression in embryo nourishing tissues endosperm and placenta in flowering plants and mammals, respectively. Seeds are formed with double fertilization in flowering plants and the endosperm has a 3n chromosome set with the contribution of 2 maternal and 1 paternal genome. Any deviation from this ratio (2m+1p) results in seed abortion in many species, however, apomict species modify their gametogenesis or fertilization to survive. Boechera divaricarpa is a diploid apomict plant species that can produce seeds with a 4m:1p parental genome ratio in endosperm and produce viable seeds. SUVH7, on the other hand, is a histone methyltransferase that has a catalytic SET domain responsible for epigenetic control of gene expression. In this study, we characterized the structures of the SUVH7 gene and compared the mRNA levels of SUVH7 in diploid apomict and sexual Boechera spp. in unopened immature buds and manually pollinated siliques representing the -pre and -post pollination stages, respectively. The expression level of SUVH7 in apomict B. divaricarpa has reached the max level 48 hours later following pollination, while in sexual B. stricta its expression level has dramatically decreased. Therefore, our study suggests the importance of epigenetic reprogramming in apomicts during seed development since chromatin marks via SUVH7 are commonly associated with the activation of transcription in plants.

New facts about callose events in the young ovules of some sexual and apomictic species of the Asteraceae family.

Callose (β-1,3-glucan) is one of the cell wall polymers that plays an important role in many biological processes in plants, including reproductive development. In angiosperms, timely deposition and degradation of callose during sporogenesis accompanies the transition of cells from somatic to generative identity. However, knowledge on the regulation of callose biosynthesis at specific sites of the megasporocyte wall remains limited and the data on its distribution are not conclusive. Establishing the callose deposition pattern in a large number of species can contribute to full understanding of its function in reproductive development. Previous studies focused on callose events in sexual species and only a few concerned apomicts. The main goal of our research was to establish and compare the pattern of callose deposition during early sexual and diplosporous processes in the ovules of some Hieracium, Pilosella and Taraxacum (Asteraceae) species; aniline blue staining technique was used for this purpose. Our findings indicate that callose deposition accompanies both meiotic and diplosporous development of the megaspore mother cell. This suggests that it has similar regulatory functions in intercellular communication regardless of the mode of reproduction. Interestingly, callose deposition followed a different pattern in the studied sexual and diplosporous species compared to most angiosperms as it usually began at the micropylar pole of the megasporocyte. Here, it was only in sexually reproducing H. transylvanicum that callose first appeared at the chalazal pole of the megasporocyte. The present paper additionally discusses the occurrence of aposporous initial cells with callose-rich walls in the ovules of diploid species.

The Effectiveness of the Sexual Reproduction in Selected Clonal and Nonclonal Species of the Genus Ranunculus.

Simple SummaryThe genus Ranunculus (buttercup) includes over 600 species, some of which are endangered, e.g., Illyrian Buttercup. Knowledge of the reproductive biology of such species may be crucial for conservation action. For this purpose, six species with different reproduction modes (nonclonal reproducing sexually by seeds only, clonal propagating by seeds and additionally vegetatively and apomictic) were observed. Selected features related to the efficiency of sexual reproduction were described: pollen viability, number of fruit set, seed viability and germination. It has been shown that in clonal species, which include the Illyrian Buttercup, the efficiency of sexual reproduction is lower compared to nonclonal species. The results will support conservation action taken for this species.Generative processes have been evaluated in six European buttercup species in order to verify the hypothesis that the reproduction efficiency of clonal species is lower than that of nonclonal ones. The study covered common species (Ficaria verna, Ranunculus auricomus, R. bulbosus, R. cassubicus, R. lanuginosus) and the endangered R. illyricus. The following properties have been assessed: pollen viability (staining method), pollen grain germination and the pollen-tube elongation in pistil tissues (fluorescence microscopy), seed formation efficiency, seed viability (tetrazolium test) and germination ability by introducing factors interrupting dormancy (low temperature and gibberellin application). Additionally, the pistil morphology was documented for R. bulbosus, R. illyricus and R. cassubicus using SEM techniques. It was demonstrated that the reproductive efficiency, expressed as the production of viable seeds able to germinate, was significantly higher in the species reproducing sexually (especially in R. lanuginosus) compared to the clonal ones. However, the complexity observed leads to separation of an additional group (cluster) of apomictic species: R. auricomus and R. cassubicus, distinguished by the lowest pollen viability and a low ability of the seeds to germinate. In the vegetatively reproducing R. illyricus, the seed formation efficiency was just 13.2% despite the having highest number of pistils in its flowers. The developed seeds of this species observed in our experiment were viable, but in general effective methods to stimulate their germination have not been proposed yet. Here, the first comparative study concerning the biology of sexual reproduction of R. illyricus is presented in the context of its decreasing distribution in natural habitats.