An adventurous journey toward and away from fern apomixis: Insights from genome size and spore abortion patterns.

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.

Interploidy gene flow via a 'pentaploid bridge' and ploidy reduction in Cystopteris fragilis fern complex (Cystopteridaceae: Polypodiales).

KEY MESSAGE: Our results indicate the existence of interploidy gene flow in Cystopteris fragilis, resulting in sexual triploid and diploid gametophytes from pentaploid parents. Similar evolutionary dynamics might operate in other fern complexes and need further investigation. Polyploidization and hybridization are a key evolutionary processes in ferns. Here, we outline an interploidy gene flow pathway operating in the polyploid Cystopteris fragilis complex. The conditions necessary for the existence of this pathway were tested. A total of 365 C. fragilis individuals were collected covering representatives of all three predominant ploidy levels (tetraploid, pentaploid, and hexaploid), cultivated, had their ploidy level estimated by flow cytometry, and their spores collected. The spores, as well as gametophytes and sporophytes established from them, were analysed by flow cytometry. Spore abortion rate was also estimated. In tetraploids, we observed the formation of unreduced (tetraploid) spores (ca 2%). Collected pentaploid individuals indicate ongoing hybridization between ploidy levels. Pentaploids formed up to 52% viable spores, ca 79% of them reduced, i.e. diploid and triploid. Reduced spores formed viable gametophytes, and, in the case of triploids, filial hexaploid sporophytes, showing evidence of sexual reproduction. Some tetraploid sporophytes reproduce apomictically (based on uniform ploidy of their metagenesis up to filial sporophytes). Triploid and diploid gametophytes from pentaploid parents are able to mate among themselves, or with "normal" reduced gametophytes from the sexual tetraploid sporophytes (the dominant ploidy level in the sporophytes in this populations), to produce tetraploid, pentaploid, and hexaploid sporophytes, allowing for geneflow from the pentaploids to both the tetraploid and hexaploid populations. Similar evolutionary dynamics might operate in other fern complexes and need further investigation.

An experimental assessment of competitive interactions between sexual and apomictic fern gametophytes using Easy Leaf Area.

Premise: Few studies have explored competition in fern gametophyte populations. One limiting factor is the tedious measurement of gametophyte size as a proxy for biomass in these small plants. Here, an alternative approach of estimating the number of green pixels from photos was employed to measure the competitive interactions among apomictic and sexual Dryopteris gametophytes. Methods: We cultivated the gametophytes of two apomictic (diploid and triploid) and one sexual (tetraploid) Dryopteris species in monocultures and in two-species mixtures in the ratios 1 : 1 and 1 : 3. The total gametophyte cover of each population originating from 20 spores was assessed using Easy Leaf Area. Assessments were performed weekly between weeks 4 and 10 of cultivation. Additionally, during week 5, the cover of each species in each mixture was estimated separately. Results: We identified a positive correlation between gametophyte size and ploidy level as well as sexual reproduction. The performance of the tested species in mixtures was dependent on the competitor species identity, indicating the importance of competition between gametophytes. Discussion: The methods outlined can be used for a rapid assessment of fern gametophyte cover in large gametophyte populations. Ploidy level and reproduction type seem to play a major role in the competitive abilities of fern gametophytes, but more research is needed on this topic.

Apomictic fern fathers: an experimental approach to the reproductive characteristics of sexual, apomict, and hybrid fern gametophytes.

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.

European ferns as rich sources of antioxidants in the human diet.

Wild edible plants have become an attractive variation of the human diet, especially in East Asia, North America, and Oceania. However, their potential in nutrition is only rarely considered in Europe. This study aims to reveal the nutritional and antioxidant potential of mature fern leaves from 13 families grown in Europe. We found that most of the examined fern species displayed a high antioxidant capacity, exceeding 0.5 g Trolox equivalent per gram of extract dry weight in ORAC assay and reaching IC50 values lower than 30 µg·mL-1 in DPPH assay (with the value for Trolox 7 µg·mL-1). Most of the species also appeared to be a good source of carotenoids, especially of lutein (205 µg·g-1 DW on average) and ß-carotene (161 µg·g-1 DW on average) when compared to the reference leafy vegetables spinach and rocket. A cytotoxicity test using ovine hepatocytes showed a non-toxicity effect of fern leaf extracts.

Nutritional and Antioxidant Potential of Fiddleheads from European Ferns.

Ferns are part of the diet and traditional medicine in East Asia, North America, and Oceania, however, their importance has been forgotten in Europe. Here, the nutritional and antioxidant potential of young fern fronds (fiddleheads) of eight families were studied. Most of the tested fern species excelled in high antioxidant capacity when compared to the reference leafy vegetables spinach and rocket. On average, the total phenol content reached 220 mg·g-1 of extract dry weight for all fiddleheads, and 15 out of 24 tested species exceeded 1 g Trolox equivalent per gram of extract dry weight in Oxygen Radical Absorbance Capacity (ORAC) assay. On the other hand, fiddleheads contained a comparable amount of carotenoids and ascorbic acid with the reference vegetables. In the case of fatty acid composition, fiddleheads contained especially high amounts of essential omega-3 (n3) and omega-6 (n6) polyunsaturated fatty acids with a beneficial n6/n3 ratio. The n6/n3 ratio in all tested species was between 2 and 6.4, whereas the ratio in the reference vegetables was below 0.4. All in all, fiddleheads from European ferns are a rich source of valuable antioxidants and essential fatty acids with a desirable n-6/n-3 ratio and may thus form an alternative source of these compounds, especially for those people not consuming fish and fish products.

Insights into the evolutionary history and widespread occurrence of antheridiogen systems in ferns.

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.

Asymmetric hybridization in Central European populations of the Dryopteris carthusiana group.

PREMISE: Hybridization is a key process in plant speciation. Despite its importance, there is no detailed study of hybridization rates in fern populations. A proper estimate of hybridization rates is needed to understand factors regulating hybridization. METHODS: We studied hybridization in the European Dryopteris carthusiana group, represented by one diploid and two tetraploid species and their hybrids. We sampled ~100 individuals per population in 40 mixed populations of the D. carthusiana group across Europe. All plants were identified by measuring genome size (DAPI staining) using flow cytometry. To determine the maternal parentage of hybrids, we sequenced the chloroplast region trnL-trnF of all taxa involved. RESULTS: We found hybrids in 85% of populations. Triploid D. ×ambroseae occurred in every population that included both parent species and is most abundant when the parent species are equally abundant. By contrast, tetraploid D. ×deweveri was rare (15 individuals total) and triploid D. ×sarvelae was absent. The parentage of hybrid taxa is asymmetric. Despite expectations from previous studies, tetraploid D. dilatata is the predominant male parent of its triploid hybrid. CONCLUSIONS: This is a thorough investigation of hybridization rates in natural populations of ferns. Hybridization rates differ greatly even among closely related fern taxa. In contrast to angiosperms, our data suggest that hybridization rates are highest in balanced parent populations and support the notion that some ferns possess very weak barriers to hybridization. Our results from sequencing cpDNA challenge established notions about the correlation of ploidy level and mating tendencies.

Widespread co-occurrence of multiple ploidy levels in fragile ferns (Cystopteris fragilis complex; Cystopteridaceae) probably stems from similar ecology of cytotypes, their efficient dispersal and inter-ploidy hybridization.

BACKGROUND AND AIMS: Polyploidy has played an important role in the evolution of ferns. However, the dearth of data on cytotype diversity, cytotype distribution patterns and ecology in ferns is striking in comparison with angiosperms and prevents an assessment of whether cytotype coexistence and its mechanisms show similar patterns in both plant groups. Here, an attempt to fill this gap was made using the ploidy-variable and widely distributed Cystopteris fragilis complex. METHODS: Flow cytometry was used to assess DNA ploidy level and monoploid genome size (Cx value) of 5518 C. fragilis individuals from 449 populations collected over most of the species' global distributional range, supplemented with data from 405 individuals representing other related species from the complex. Ecological preferences of C. fragilis tetraploids and hexaploids were compared using field-recorded parameters and database-extracted climate data. KEY RESULTS: Altogether, five different ploidy levels (2x, 4x, 5x, 6x, 8x) were detected and three species exhibited intraspecific ploidy-level variation: C. fragilis, C. alpina and C. diaphana. Two predominant C. fragilis cytotypes, tetraploids and hexaploids, co-occur over most of Europe in a diffuse, mosaic-like pattern. Within this contact zone, 40 % of populations were mixed-ploidy and most also contained pentaploid hybrids. Environmental conditions had only a limited effect on the distribution of cytotypes. Differences were found in the Cx value of tetraploids and hexaploids: between-cytotype divergence was higher in uniform-ploidy than in mixed-ploidy populations. CONCLUSIONS: High ploidy-level diversity and widespread cytotype coexistence in the C. fragilis complex match the well-documented patterns in some angiosperms. While ploidy coexistence in C. fragilis is not driven by environmental factors, it could be facilitated by the perennial life-form of the species, its reproductive modes and efficient wind dispersal of spores. Independent origins of hexaploids and/or inter-ploidy gene flow may be expected in mixed-ploidy populations according to Cx value comparisons.

Between sexual and apomictic: unexpectedly variable sporogenesis and production of viable polyhaploids in the pentaploid fern of the Dryopteris affinis agg. (Dryopteridaceae).

BACKGROUND AND AIMS: In ferns, apomixis is an important mode of asexual reproduction. Although the mechanisms of fern reproduction have been studied thoroughly, most previous work has focused on cases in which ferns reproduce either exclusively sexually or exclusively asexually. Reproduction of ferns with potentially mixed systems and inheritance of apomixis remains largely unknown. This study addresses reproduction of the pentaploid Dryopteris × critica, a hybrid of triploid apomictic D. borreri and tetraploid sexual D. filix-mas. METHODS: Spore size, abortion percentage and number of spores per sporangium were examined in pentaploid plants of D. × critica grown in an experimental garden. The sporangial content of leaf segments was cultivated on an agar medium, and DNA ploidy levels were estimated by DAPI flow cytometry in 259 gametophytes or sporophytes arising from the F2 generation of the pentaploid hybrid. KEY RESULTS: The hybrid is partly fertile (89-94% of aborted spores) and shows unstable sporogenesis with sexual and apomictic reproduction combined. The number of spores per sporangium varied from approx. 31 to 64. Within a single sporangium it was possible to detect formation of either only aborted spores or various mixtures of aborted and well-developed reduced spores and unreduced diplospores. The spores germinated in viable gametophytes with two ploidy levels: pentaploid (5x, from unreduced spores) and half of that (approx. 2·5x, from reduced spores). Moreover, 2-15% of gametophytes (both 2·5x and 5x) formed a viable sporophyte of the same ploidy level due to apogamy. CONCLUSIONS: This study documents the mixed reproductive mode of a hybrid between apomictic and sexual ferns. Both sexual reduced and apomictic unreduced spores can be produced by a single individual, and even within a single sporangium. Both types of spores give rise to viable F2 generation gametophytes and sporophytes.

Continuous morphological variation correlated with genome size indicates frequent introgressive hybridization among Diphasiastrum species (Lycopodiaceae) in Central Europe.

Introgressive hybridization is an important evolutionary process frequently contributing to diversification and speciation of angiosperms. Its extent in other groups of land plants has only rarely been studied, however. We therefore examined the levels of introgression in the genus Diphasiastrum, a taxonomically challenging group of Lycopodiophytes, using flow cytometry and numerical and geometric morphometric analyses. Patterns of morphological and cytological variation were evaluated in an extensive dataset of 561 individuals from 57 populations of six taxa from Central Europe, the region with the largest known taxonomic complexity. In addition, genome size values of 63 individuals from Northern Europe were acquired for comparative purposes. Within Central European populations, we detected a continuous pattern in both morphological variation and genome size (strongly correlated together) suggesting extensive levels of interspecific gene flow within this region, including several large hybrid swarm populations. The secondary character of habitats of Central European hybrid swarm populations suggests that man-made landscape changes might have enhanced unnatural contact of species, resulting in extensive hybridization within this area. On the contrary, a distinct pattern of genome size variation among individuals from other parts of Europe indicates that pure populations prevail outside Central Europe. All in all, introgressive hybridization among Diphasiastrum species in Central Europe represents a unique case of extensive interspecific gene flow among spore producing vascular plants that cause serious complications of taxa delimitation.

Species boundaries and frequency of hybridization in the Dryopteris carthusiana (Dryopteridaceae) complex: A taxonomic puzzle resolved using genome size data.

PREMISE OF THE STUDY: Genome duplication and interspecific hybridization are important evolutionary processes that significantly influence phenotypic variation, ecological behavior, and reproductive biology of land plants. These processes played a major role in the evolution of the Dryopteris carthusiana complex. This taxonomically intricate group composed of one diploid (D. expansa) and two allotetraploid (D. carthusiana and D. dilatata) species in Central Europe. Overall phenotypic similarity, great plasticity, and the incidence of interspecific hybrids have led to a continuous dispute concerning species circumscription and delimitation. • METHODS: We used flow cytometry and multivariate morphometrics to assess the level of phenotypic variation and the frequency of hybridization in a representative set covering all recognized species and hybrids. • KEY RESULTS: Flow cytometric measurements revealed unique genome sizes in all species and hybrids, allowing their easy and reliable identification for subsequent morphometric analyses. Different species often formed mixed populations, providing the opportunity for interspecific hybridization. Different frequencies of particular hybrid combinations depended primarily on evolutionary relationships, reproductive biology, and co-occurrence of progenitors. • CONCLUSIONS: Our study shows that genome size is a powerful marker for taxonomic decisions about the D. carthusiana complex and that genome size data may help to resolve taxonomic complexities in this important component of the temperate fern flora.