In spite of its relatively infrequent occurrence, long distance colonization is of disproportionate importance to species range expansions [e.g. [1, 2]]. Long-distance colonization requires plant species' to possess a distinct set of capabilities, not only related to the dispersal of propagules, but also to plant and population establishment upon arrival. This involves diaspore characteristics, plant ontogenetic and morphological traits, as well as reproductive strategies. Genotypes possessing these capabilities will have a selective advantage over other genotypes when colonizing new and distant habitats. This advantage is becoming more important in a world increasingly under the pressure of climate change and fragmentation of natural habitats .
Various studies on plants and animals have shown that individuals with higher dispersal capacities tend to be found with greater frequency towards species' range limits [4, 5] and that these enhanced capacities tend to have a genetic basis . Likewise, inbreeding rates often increase towards range margins . This might partly be due to genetic isolation and small population sizes , but can also be explained by reproductive assurance . As colonization of vacant patches near a species' range limits will often depend on rare events of diaspore arrival through long-distance dispersal [e.g. ], mate limitation is likely high  and colonization success may strongly depend on self-fertilization. For this reason, Baker [11, 12] suggested that establishment of selfing individuals will be strongly favoured after long-distance dispersal. Baker's law  states that long-distance colonization may therefore result in selection for individuals with high self-fertilization potential. As a result, plants in young populations near a species' range limit sometimes show relatively low self-incompatibility . However, whether such selection occurs and how long this effect remains visible in the populations after initial colonization, depends on the dominant mating strategy, as well as the intraspecific variation in mating strategy present in the species investigated [e.g. ]. Selection for genotypes capable of self-fertilization will not occur in species that lack any intraspecific variation in mating strategy. Moreover, the overrepresentation of selfing genotypes may be reduced with time since colonization as a result of inbreeding depression : the reduced success of inbred progeny due to the expression of genetic load (i.e. recessive deleterious alleles).
In ferns, which alternate between two free-living generations (gametophyte and sporophyte), sexual reproduction takes place on the gametophyte. After a spore has reached a suitable habitat patch and has germinated, fertilization of the gametophyte is required for sporophyte establishment . In homosporous ferns, gametophytes have the potential to become male, female or bisexual. Sexual status typically varies between individuals and depends both on genetic factors and environmental conditions . The possibility of producing hermaphroditic gametophytes allows for self-fertilization of a single gametophyte (i.e. intragametophytic selfing ). This potential is of particular importance for fern colonization, as very limited gamete dispersal distances result in strong mate limitation. This may strongly limit options for cross-fertilization as long as no local spore sources are present [18, 19]. Long-distance colonization thus might be primarily dependent on reproduction via single spores, through intragametophytic selfing . This type of reproduction represents an extreme case of inbreeding. Fern gametophytes contain only half the number of sets of chromosomes of the somatic cells of the sporophyte. As gametophytes of diploid ferns thus have only a single copy of each chromosome, intragametophytic selfing in diploid ferns produces sporophytes that are homozygous at all loci. This results in the direct expression of any recessive deleterious alleles in the sporophytes produced, which may severely affect the fitness of inbred sporophytes . Gametophytes of polyploids possess multiple copies of each gene. Therefore, both the gametophytes and the gametes they produce may contain multiple alleles per locus. Even after intragametophytic selfing, recessive deleterious alleles may therefore be masked by other alleles in the sporophyte, making the effects of inbreeding depression less pronounced. For that reason, polyploid ferns are generally assumed to show enhanced survival of inbred progeny, and higher population inbreeding rates .
Fern mating systems can be studied experimentally using breeding experiments . Such experiments compare sporophyte production by obligate intragametophytic selfing on isolated gametophytes with sporophyte production in paired cultures, in which case also intergametophytic crossing is possible (or intergametophytic selfing, if the second gametophyte originates from the same parent sporophyte ). Ferns generally seem to lack genetic self-incompatibility mechanisms , but unsuccessful self-fertilization may be caused by a failure of the gametophyte to become bisexual, unsuccessful transport of spermatozoids to the female reproductive organs, or the presence of genetic load. Species differences in sex ratios, gametophyte morphology and genetic load may therefore result in different types of mating strategies. Together with studies on observed genetic variation in fern populations, past breeding experiments suggested that the mating strategies employed by fern species vary in a bimodal way: some species reproduce mainly by self-fertilization and others rely on obligate intergametophytic crossing [17, 23–26]. However, some species do show mixed mating systems [27, 28], and by now several studies have indicated that mating systems may vary greatly even between different genotypes within species [18, 29, 30]. This intraspecific variation is in line with the large variation in inbreeding rates observed among sites in population genetic studies [e.g. [31, 32]]. However, as breeding experiments with multiple genotypes are very laborious, intraspecific variation in mating strategy has only been assessed for a few species. Due to this lack of data, it remains largely unknown to what extent selfing genotypes are also present in species previously described as typical outcrossers, and how intraspecific variation in mating strategy differs between diploid and polyploid species. Therefore, it is also unclear to what extent selection for selfing genotypes, sensu Baker [11, 12], is a widespread phenomenon in ferns.
In this study, we simultaneously investigated inter-and intraspecific variation in mating strategy in four temperate fern species, including two diploid and two allotetraploid species. We performed breeding experiments on several genotypes per species, determining the success of sporophyte production at different levels of inbreeding. In this way, we tested whether intraspecific variation in mating system varied between species, whether genotypes with a high capacity to self-fertilize are present in all four species, and whether selfing capacities and overall mating strategies differed between diploid and polyploid species.
Most genotypes used were derived from young populations in the Kuinderbos, a planted forest on Dutch polder land recently reclaimed from the sea. As the four investigated species are all rare in the Netherlands, with nearest source populations located 30-350 km away, the populations in the Kuinderbos must have established following long-distance dispersal [33, 34]. A population genetic study (G.A. de Groot, unpublished results) suggested that most populations are the result of independent colonization events. Because such populations have likely established from single spores , we predicted that the sampled genotypes might have relatively high capacity for self-fertilization. This capacity was, however, expected to be lower for the diploid than for the polyploid species. We found surprisingly high selfing capacities for all Kuinderbos genotypes of all four species, both compared to results for a few additional genotypes from less isolated populations and compared to results from previous studies on the same species. Here, we interpret our results in the light of Baker's law, and suggest that selection for selfing genotypes may occur across fern species with different ploidy levels.