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.

• Keywords
• Apomixis, Cystopteris, Diplospores, Ferns, Flow cytometry, Gametophytes, Interploidy gene flow, Mixed mating, Ploidy reduction, Sporogenesis,
• Publication type
• Journal Article MeSH

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.

• Keywords
• Dryopteris, apomixis, competition, monoculture, pteridophyte, spore, wood fern,
• Publication type
• Journal Article 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

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.

• Keywords
• Cystopteris fragilis, Bladder ferns, Cx value, contact zone, cytotype coexistence, ecological preferences, flow cytometry, genome size, ploidy distribution, pteridophytes,
• MeSH
• Ecology MeSH
• Hybridization, Genetic MeSH
• Ferns * MeSH
• Humans MeSH
• Ploidies MeSH
• Polyploidy MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Geographicals
• Europe MeSH