DOI: 10.5061/dryad.np5hqbzqc
title: Data from: Outcrossing mating system of the early-divergent moonwort fern (Botrychium lunaria, Ophioglossaceae) revealed in the European Alps

description: Premise of the Research. Vascular plants depend on sexual
 recombination for generating new genetic variability to meet environmental
 needs. Nevertheless, members of the early-divergent fern
 genus Botrychium (Ophioglossaceae) typically maintain
 gametophytic selfing and show strong inbreeding within populations. To
 explain this evolutionary anomaly, the existence of previous or current
 but undiscovered outcrossing, genetically rich, precursors of the existing
 genetically depauperate taxa has been hypothesized. 
 Methodology. Using allele expression at thirteen independently
 assorting enzyme loci, we compared allelic diversity and levels of
 heterozygosity in 471 Botrychium lunaria individuals
 across sixteen populations in the Alps and Jura Mountains of Switzerland.
 We examined habitat characteristics influencing mating systems and
 investigated population genetic structure based on a discriminant analysis
 of principal components and a graph-theoretic framework. We tested the
 pattern of isolation by distance and explored the biological processes
 favoring or limiting spore dispersal at a regional scale. Pivotal
 Results. We found high genetic diversity within and among
 populations, and similar observed (HO) and expected heterozygosity (HS)
 across loci (HO = 0.020-0.450 and HS =
 0.040-0.590, respectively). Estimates of HO(0.050-0.272) were
 lower than HS (0.144-0.305) across individuals,
 indicating a weak deficit in heterozygotes. Mean values of departure (D)
 from expected heterozygosity were close to what would be expected with
 random mating (DL = 0.061 and DP = 0.059 across loci and
 populations, respectively). The mean inbreeding coefficient was low
 (FIS = 0.247) and the overall genetic differentiation was
 moderate (global FST = 0.083).
 Conclusions. Together, the discriminant analysis of principal
 components and the Population Graph revealed a complex population genetic
 structure and a weak genetic differentiation with substantial gene flow
 among most populations. One population (Arosa), sampled from a site
 recently released from permafrost, demonstrated probable founder effect
 not correlated with geographical isolation. The topology of
 the Population Graph identified three subgroups with several key
 populations (BGU, CHL, GSB, SBE, and VRO) that maintained genetic
 connectivity of populations from distant locations.
description: Allozyme data were generated following the procedures of Stensvold & Farrar (2017). As soon as possible after collection, 1 cm of tissue was cut from the base of the leaf stalk and crushed in a buffer solution containing phosphate-polyvinylpyrrolidone (Cronn et al. 1997). The grindate was stored for up to 12 months in microcentrifuge tubes at –80°C until electrophoresis was conducted. Enzyme electrophoresis was performed in horizontal 12% starch gel using the three buffer systems 7, 9, and 11 from Soltis et al. (1983) to reveal allelic variants at 22 loci of ten enzyme systems by differentiating gel migration patterns. Thus, loci of those enzyme systems were resolved for aspartate aminotransferase (AAT) and triose-phosphate isomerase (TPI) with buffer system 7, for malate dehydrogenase (MDH), phosphoglucomutase (PGM), phosphogluconate dehydrogenase (PGD), and phospho-glucoisomerase (PGI) with buffer system 9 and, for aconitase (ACN), diaphorase (DIA), isocitrate dehydrogenase (IDH), and shikimate dehydrogenase (SKDH) with buffer system 11. The stain recipes used followed Soltis et al. (1983). Allele letter codes (i.e., scoring) were assigned with reference to alleles previously determined for <i>Botrychium</i> taxa of the <i>B. lunaria</i> complex by Stensvold & Farrar (2017). Consistency of scores was checked by multiple runs of the same specimens when necessary. Allele variants for each locus were numbered, assigning the lowest number to the fastest (most anodal) migrating allele. In analyses we removed rare alleles (population frequencies &lt;0.05) to avoid artificial inflation of polymorphism estimates due to experimental error as recommended by Hartl & Clark (1998) and excluded uninformative loci that were monomorphic across all populations. Loci with missing scores in more than 30% of samples were excluded.