We know little about the underlying genetic control of phenotypic patterns of seed traits across large-scale geographic and environmental gradients. Such knowledge is important for understanding the evolution of populations within species and for improving species conservation. Therefore, to test for genetic variation in P. lanceolata, we made reciprocal crosses between northern and southern genotypes that span the species’ range in Europe. Results provide evidence of transgenerational genetic effects on seed mass and germination timing. Northern mothers produced larger seeds with delayed germination, in contrast to southern mothers, which produced smaller seeds with accelerated germination. Maternal latitude affected both the seed coat, solely maternal tissue, and embryo/endosperm tissues. Thus, latitudinal variation in seed size and germination timing can be explained, in part, by the direct influence of maternal genotype, independent of zygotic genes that parents pass directly to the embryo and endosperm. Data suggest that researchers exploring the existence and evolution of large-scale geographic variation within species test for transgenerational genetic effects. Also, data suggest that transgenerational control of seed traits should be considered when developing procedures designed to facilitate species conservation and restoration.

Our two experiments derive from a QTL experiment in which Marshall et al. (2020) [92] explored the genetic architecture of differences in thermal plasticity in flower color and flowering time between northern and southern European populations. Because we used F1 seeds from this experiment, we summarize aspects of the QTL experiment and refer readers to Marshall et al. (2020) [92] and to an earlier experiment describing the latitudinal variation in thermal plasticity in flower color [97] for more details. The northern and southern parents used in the QTL study displayed high and low thermal plasticity, respectively, and represented plasticity extremes found in a sample of 29 European P. lanceolata populations. Parents in the QTL study were themselves progeny of genotypes that had been collected from wild European populations differing in thermal regime and duration of the growing season (Fig. 2, See also [97]). To reduce maternal environmental effects, Lacey et al. (2010) [97] had induced genotypes to flower and set seed while keeping the wild populations separated but in similar controlled environments. Plants grew vegetatively in a greenhouse until flowering began, at which time populations were isolated in growth chambers and separate greenhouse rooms set at 22oC, 16-h day/17oC, 8-h night to allow for random within-population wind pollination and seed production. In 2012, Marshall et al. (2020) [92] reciprocally crossed combinations of genotypes derived from northern and southern source populations (i.e., hereafter referred to as the parental generation) to produce F1 seeds (Fig. 2). Multiple clones of parental genotypes were used to produce F1 seeds. For all crosses, the single growth chamber was set at 20°C, 16-hr day/15°C, 8-hr night. Thus, the seeds that we examined were products of two generations of crosses made in similar environments, which further reduced remnant parental environmental effects that might have persisted after the first generation. F1 seeds were harvested, counted and stored at room temperature in the lab for several months, which allowed time for after-ripening to be completed. Then we sampled seeds for the two experiments, described below. For both, we chose seeds that were brown and shiny and avoided seeds that were black and flat, which indicated that the seeds had been aborted.

Experiment 1: Five northern parental genotypes (one per population) were reciprocally crossed with five southern genotypes (one from a French population and four from an Italian population). The four genotypes from the Italian population were descended from different grandmothers. For each reciprocal cross, we randomly sampled 20 seeds per clonal cross (1-3 clonal cross per reciprocal) and weighed seeds, each to the nearest 0.001 mg.

Experiment 2: We sampled 4 pairs of reciprocal crosses again, but this time we collected data on total seed mass, coat mass, endosperm/embryo mass, and days to germination. Fifteen seeds per reciprocal cross were selected randomly and independently of clone. After weighing each seed individually per reciprocal cross, we placed 3 seeds in each of 5 petri dishes lined with filter paper that was saturated with water. The petri dishes were placed in a growth chamber set at 20°C, 16-hr day/15°C, 8-hr night, and water was added as needed to keep the filter paper moist. Seeds were checked daily. We recorded the day of appearance of a radicle protruding from the seed coat. As germination continued, we collected the seed coats, which drop off after germination. Coats were dried and stored at room temperature for a week after all germination had ceased. All seeds germinated within 7 days from the day of placement in the growth chamber.

Instructions for reading and understanding the datafiles are in the README document.