Plant material

Potentilla reptans L. is a stoloniferous perennial herb, which occurs in meadows and river banks as well as disturbed environments, such as roadsides and pastures52. It grows stolons with rooted ramets, which normally stay connected to the mother plant for one season52 and has been shown to have high levels of clonal integration53. The ramets are rosettes that form indeterminate number of leaves whose height is regulated by petiole length and vertical inclination. Leaf height has been shown to increase with that of neighbouring plants36, 47, while leaf number has been shown to decrease under shade54, 55.

Twenty P. reptans cuttings from different sites around Tübingen, Germany were collected in December 2013. To relax environmentally-induced maternal effects, the cuttings were clonally propagated for one generation under common-garden conditions at a field site in Tübingen University, where they were individually planted in 1 L pots and covered with light fabric organza during winter.

Experimental setup

In May 2014, four newly grown ramets were severed from each P. reptans mother plant. Each ramet was then planted separately in the centre of a 12 L pot (30 cm diameter) filled with local topsoil (Bischoff GmbH & Co. KG, Rottenburg am Neckar, Germany). Individual ramets, one ramet per genotype, were assigned to one of four light-competition treatments simulating different height and density of neighbours. Light competition was simulated using transparent green plastic filters, which mimic vegetative shade in both light transmission levels and R:FR ratios (122 Fern green, Lee filters, CA, USA)23, 36. One centimetre-wide filter strips were used to create a grid of two concentric cylinders of 15 and 30 cm in diameter (Fig. 4a–f). The use of two cylinders rather than a single one provided a better simulation of light competition that could be experienced by clonal plants as they expand horizontally. To simulate vegetation through which plants could grow laterally, the filter strips were positioned 0.5 cm apart. Short and tall neighbours were simulated using 15- and 50-cm-long filter strips, respectively (Fig. 4a–f). Sparse neighbours were simulated with alternating green and clear (130 clear, Lee filters, CA, USA) strips (Fig. 4a, c, e), while dense neighbours were simulated using green strips only (Fig. 4b, d, f). This experiment resulted in a total of 80 plants (20 genotypes × 2 density treatments × 2 height treatments). The sample size (n = 20) was chosen based on previous studies where developmental plasticity in P. reptans in response to light competition was shown for a smaller sample of ca. 10 genotypes36, 55.

The filters were set around the plants following transplantation and the pots were placed on benches at a greenhouse in Tübingen University, within a distance of 50 cm between pots, to prevent shading effects among neighbouring plants. The plants were arranged in blocks according to genotype and assigned random numbers to conceal their identity during variable measurements. Photosynthetically active radiation (PAR) was measured on September 2015 for a subset of pots (seven per treatment) at four heights from the soil (2, 6, 10 and 14 cm) using a quantum light metre (LI-250, LI-COR Inc., Lincoln, NE, USA). PAR differed among treatments (ANOVA: F 3,96 = 37.087, P < 0.001), height from soil (ANOVA: F 3,96 = 18.167, P < 0.001) and their interaction (ANOVA: F 9,96 = 4.883, P < 0.001). At a height of 2–6 cm, which is leaf-lamina height at the beginning of the experiment, PAR was higher in the short-sparse compared to the tall-dense treatment, while the short-dense and tall-sparse treatments had similar intermediate PAR levels (Fig. 5a). However, the two short treatments were characterised by a gradient of increased PAR with increasing height, while PAR levels at the two tall treatments remained low (Fig. 5a). In addition to PAR levels, red to far-red ratio (R:FR) was measured in July 2017 for a subset of pots (eight per treatment) at the same four heights using a FieldSpec 4 Standard-Res Spectroradiometer (ASD Inc., Longmont, CO, USA). R:FR differed among treatments (ANOVA: F 3,112 = 53.949, P < 0.001), height from soil (ANOVA: F 3,112 = 4.327, P = 0.006) and their interaction (ANOVA: F 9,112 = 2.018, P = 0.044). As for PAR levels, R:FR was highest in the short-sparse treatment and lowest in the tall-dense treatment (Fig. 5b). Here however, only the short-dense treatment showed a gradient of increased R:FR with increasing height (Fig. 5b). Hence, in summary, each of the treatments exhibited a unique light environment, which was expressed in light intensity, light quality or both, and/or a gradient in these light characteristics.

Fig. 5 Light characteristics in the different treatments simulating vegetative shade. Values are means ± SEM of photosynthetically active radiation (PAR; a) and red to far-red ratio (R:FR; b) at different heights within the short (circles and dashed lines), tall (triangles and solid lines) sparse (bright green) and dense (dark green) treatments. Different letters indicate statistically significant differences between heights within a treatment, estimated using the least significant difference (LSD) test with the false discovery rate correction59 following an ANOVA in a (ANOVA, height from soil: P < 0.001, n = 7) and b (ANOVA, height from soil: P = 0.006, n = 8) Full size image

Measured variables

Ramet performance in response to the different treatments was estimated from the number of newly produced leaves per ramet, i.e. number of leaves at the end of the experiment minus number of leaves at its onset. Leaf number has been shown to provide an appropriate non-destructive estimate of plant size in P. reptans 54, and was also found to highly correlate with ramet biomass in a separate experiment (Supplementary Fig. 1a). The numbers of newly produced leaves in one ramet in the short-sparse, short-dense and tall-dense treatments were lost due to technical problems, resulting in a sample size of 19 per treatment.

Measurements of the plastic responses of P. reptans were carried out in August 2014, 9 weeks after the onset of the experiment. These measurements included petiole length as well as plant height, which was estimated as the vertical distance between the highest leaf tip and the soil surface. Vertical inclination in response to competition was evaluated as the ratio between plant height and its diameter, i.e. the maximum distance between the two furthermost leaf tips. This ratio was chosen because it proved easier to measure within the shading apparatus compared to petioles angles, and due to its high correlation with the latter (Supplementary Fig. 1b). Plant height in one ramet in the short-sparse treatment was lost due to technical problems, resulting in a sample size of 19 for this treatment.

Shade tolerance was estimated with specific leaf area, i.e. the ratio between lamina area and its dry weight. To that end, the two biggest laminas per ramet were harvested and photographed, and their images were used to quantify mean lamina area with the ImageJ software56. Laminae’s biomass was measured following oven drying them in 70 ˚C for 3 days. Lateral clonal growth was estimated by measuring total stolon length per plant as well as mean internode length of the stolons.

All variables were measured in the pots prior to the removal of the filters so as to not disrupt plant architecture, except for lamina area and biomass. For the latter variables, samples were identified according to randomly assigned numbers rather than treatment names to blind the investigator to treatment identity. All collected data are available in the Supplementary Data.

Data analysis

The responses of P. reptans to the height and density of simulated neighbours expressed by their number of newly produced leaves, petiole length, height-per-diameter ratio, specific leaf area, total stolon length and mean internode length were examined using a generalised linear-mixed model with filter height and density as fixed factors and plant genotype as a random factor. The number of newly produced leaves, petiole length, height-per-diameter ratio and specific leaf area were analysed with a normal probability distribution with an identity link function. Total stolon length and internode length were analysed with a Gamma probability distribution with a log link function. To account for potential differences in total stolon length due to ramet size57, 58, the number of leaves per ramet at the end of the experiment was used as a covariate (after confirming the assumption of homogeneity of slopes). Post hoc pairwise comparisons between treatments were performed using false discovery rate correction for multiple tests59, 60. These statistical analyses were performed using PASW 18 (SPSS).

In addition to the univariate analyses, a multivariate approach was employed to evaluate the complete array of plastic responses displayed by P. reptans under the different treatments. Partial redundancy analysis (RDA) was performed with diameter, height per diameter, leaf area, mean internode length, number of new leaves, petiole length, plant height, specific leaf area and total stolon length as response variables explained by the simulated neighbour treatments after removing the effect of covariates (number of leaves at the end of the experiment as an estimate of plant size and genotype). To account for different measuring units, traits were centred and standardised. Monte-Carlo permutation test (n = 999) on first and second RDA axes was used with genotype as permutation block61. Furthermore, to evaluate the effect of each of the treatments independently, simple tests for each treatment were performed using the false discovery rate correction for multiple tests50, 62. The multivariate analyses were performed using CANOCO 563.

To better estimate the treatment effects on the allometric relationships between plant height and diameter as well as between total stolon length and number of leaves, these relationships and effects were analysed using the SMA regression, which is appropriate for analysing variables that have no causal relationship64. SMA was used to test for the effect of filter height and density on shifts along a common slope and on elevation (y-intercept) between slopes (after confirming the assumption of homogeneity of slopes). The traits were log-transformed prior to the analysis. These analyses were performed using the smart package in R62.

Data availability

Data analysed in this study are included in the Supplementary Information