Techniques for locating and analyzing subterranean Lycopodium and Diphasiastrum gametophytes in the field.

Homosporous club mosses have an archaic life cycle, alternating two locationally, nutritionally, and physiologically independent generations. The sexual generation of club mosses-the gametophytes (or prothallia)-are among the least researched botanical subjects. The gametophytes are responsible for not only sexual reproduction, but also the determination of recruitment of the new sporophyte generation, species habitat selection, migration, and evolution. Researchers often fail to find juvenile club moss populations and thus do not discover subterranean long-lived achlorophyllous gametophytes. To date, the gametophytes of most club moss species remain undiscovered in nature and are not scientifically documented. Almost all researchers who have previously located subterranean club moss gametophytes declared that their first find was due to luck and that subsequently the researcher's intuition plays the most important role; however, intuition and good luck are not scientific methods. In our review, we combine our knowledge with data available in the literature and discuss the following questions using a methodical approach: (1) How can we locate a subterranean club moss gametophyte population? (2) How can we extract the gametophytes? and (3) What new knowledge about club moss population development can be gained by analyzing juvenile club moss populations?

Genetic diversity and hybrid formation in Central European club-mosses (Diphasiastrum, Lycopodiaceae) - New insights from cp microsatellites, two nuclear markers and AFLP.

In Europe, the genus Diphasiastrum (Lycopodiophyta) forms a reticulate network of six diploid taxa, including three parent species (D. alpinum, D. complanatum and D. tristachyum) and three hybrids (D. × issleri, D. × oellgaardii and D. × zeilleri). It was not clear if the hybrids arose once or repeatedly, if they have reproductive competence and if backcrossing occurs. We addressed these questions by analysing 209 accessions for chloroplast microsatellites (cp), two nuclear markers (introns of the RPB and LFY genes) and AFLP. For D. complanatum we show a sexual life cycle with alternation of generations: the gametophytic DNA amount is half of that of the sporophyte. With the exception of a single accession all hybrids display one of the two parental cp haplotypes; their frequencies do not differ significantly from a 1:1 ratio. Genotypes of nuclear markers are species-specific, displaying 2/4/1 (RPB) and 1/8/1 alleles (LFY) for the three parents mentioned above; all hybrids have one allele from each parent. All three hybrid taxa apparently represent independent F1 crosses. Hybridisation occurs bidirectional; no evidence for recent backcrossing was found. Asexual reproduction via agamospory is at least rare, since AFLP showed all hybrid plants to be different.