Mating system evolution and genetic structure of diploid sexual populations of Cyrtomium falcatum in Japan

Evolution of mating systems has become one of the most important research areas in evolutionary biology. Cyrtomium falcatum is a homosporous fern species native to eastern Asia. Two subspecies belonging to a sexual diploid race of C. falcatum are recognized: subsp. littorale and subsp. australe. Subspecies littorale shows intermediate selfing rates, while subsp. australe is an obligate outcrosser. We aimed to evaluate the process of mating system evolution and divergence for the two subspecies using restriction site associated DNA sequencing (RAD-seq). The results showed that subsp. littorale had lower genetic diversity and stronger genetic drift than subsp. australe. Fluctuations in the effective population size over time were evaluated by extended Bayesian skyline plot and Stairway plot analyses, both of which revealed a severe population bottleneck about 20,000 years ago in subsp. littorale. This bottleneck and the subsequent range expansion after the LGM appear to have played an important role in the divergence of the two subspecies and the evolution of selfing in subsp. littorale. These results shed new light on the relationship between mating system evolution and past demographic change in fern species.

The C-Fern (Ceratopteris richardii) genome: insights into plant genome evolution with the first partial homosporous fern genome assembly

Ferns are notorious for possessing large genomes and numerous chromosomes. Despite decades of speculation, the processes underlying the expansive genomes of ferns are unclear, largely due to the absence of a sequenced homosporous fern genome. The lack of this crucial resource has not only hindered investigations of evolutionary processes responsible for the unusual genome characteristics of homosporous ferns, but also impeded synthesis of genome evolution across land plants. Here, we used the model fern species Ceratopteris richardii to address the processes (e.g., polyploidy, spread of repeat elements) by which the large genomes and high chromosome numbers typical of homosporous ferns may have evolved and have been maintained. We directly compared repeat compositions in species spanning the green plant tree of life and a diversity of genome sizes, as well as both short- and long-read-based assemblies of Ceratopteris. We found evidence consistent with a single ancient polyploidy event in the evolutionary history of Ceratopteris based on both genomic and cytogenetic data, and on repeat proportions similar to those found in large flowering plant genomes. This study provides a major stepping-stone in the understanding of land plant evolutionary genomics by providing the first homosporous fern reference genome, as well as insights into the processes underlying the formation of these massive genomes.

Spore Germination, Gametophyte Development and Studies of the Growth Rate of Adiantum capillus-veneris L.

Spore culture of Adiantum capillus-veneris was carried out with a view to massively raising new fernlets cultured in growth nutrient with agar and to study its growth rate. Petri-dishes B, C, D and E containing modified Moores nutrient were used with ‘A’ containing wet soil without nutrient and served as control. All the Petri-dishes were autoclaved for 15 minutes and allowed to cool before sowing sterilized spores in three replicates and maintained at pH 5.80 and 23±2 °C with cool fluorescent white light in the incubator. Spores imbibed water from the nutrient, ruptured and germination started six days after sowing with emergence of colourless, uniseriate, elongated filamentous rhizoid. Prothallial cell which was green in colour started growing perpendicularly to the rhizoid. The germination rate was scored at an interval of four days for thirty days. Various stages of growth recorded include filamentous, spatulate, prothallus and two leaved fernlets. The heart-shaped gametophyte developed between 27-60 days with 12-16 rhizoids, archegonia and antheridia. Both archegonia and antheridia were found on each gametophyte, showing the evidence of being homosporous fern. This type of gametophyte growth and development is called Drynaria type. After 8 weeks from the transfer into the garden soil, plant height and increase in the number of leaves were observed and recorded. No growth was observed in the control showed there is need for protocol in raising massive fernlets rapidly within a short time. The study showed that A. capillus-veneris could be raised easily from freshly collected spores.

Antheridiogen concentration and spore size predict gametophyte size in Ceratopteris richardii

In many plants females invest more in reproduction than males. In organisms that exhibit environmental sex determination, individuals in low-quality environments or who are slow growing are expected to develop into males. The gametophytes of Ceratopteris richardii Brongn., a homosporous fern, may develop as males or hermaphrodites. Hermaphrodites secrete a pheromone called antheridiogen that induces undifferentiated spores to develop as males. Given that induction is not 100% in the presence of antheridiogen, it is hypothesized that resources may alter C. richardii gender decisions. An experiment was undertaken to determine (i) whether spore size predicts gender, (ii) whether spore size predicts gametophyte size, (iii) whether antheridiogen negatively affects the growth of C. richardii, and (iv) whether wild-type C. richardii and him1 mutants (genetic mutants disposed to male development regardless of antheridiogen presence) behave similarly in their response to antheridiogen. Spore size was not predictive of gender but was positively related to both male and hermaphrodite gametophyte size. Antheridiogen was found to slow the growth of male and hermaphrodite gametophytes of the wild type and male gametophytes of the him1 mutant. These results are supportive of the idea that gender may be determined indirectly through antheridiogen’s effect on gametophyte growth.

Opportunistic heterotrophy in gametophytes of the homosporous fern Ceratopteris richardii

Fern gametophytes are extremely shade-tolerant, potentially existing for long periods under conditions of extreme light limitation. Many previous studies have demonstrated an increase in gametophyte growth and incidence of spontaneous transition to sporophyte morphology (apogamy) under culture on media containing exogenous sugar. However, these studies did not verify sugar uptake or quantify relative growth on media containing different sugar types. Here, we examine the extent of heterotrophy and underlying mechanisms of sugar transport in photosynthetic gametophytes of the fern Ceratopteris richardii Brongn. Exogenous sugar uptake, growth, and sugar transport were evaluated with assays of exogenous glucose depletion, experimental culture of gametophytes under different sugar and light conditions, and bioinformatic approaches. The glucose from the growth media was significantly depleted by gametophytes growing under all conditions, especially those in the dark compared with those exposed to higher light. Gametophyte area increased similarly when cultured on equimolar concentrations of either glucose or the disaccharide sucrose, likely due to preferential uptake of one of the monomers of sucrose. Although at least one gene with similarity to sucrose transporters is expressed in germinating spores, our results suggest a reliance on monosaccharide transport for exogenous sugar uptake. Glucose assimilation in both light and dark conditions constitutes nutritional opportunism and may enhance gametophyte survival in very low light.